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In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-β-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.
Author Summary
Polyketide synthases (PKSs) form a large family of multifunctional proteins involved in the biosynthesis of diverse classes of natural products. Mycobacterium tuberculosis (Mtb) exploits these polyketide biosynthetic enzymes to synthesize complex lipids, many of which are essential for its virulence. PKSs utilize two common mechanistic themes to produce these metabolites: (1) modular—in which each set of catalytic sites is used only once during the entire biosynthetic process and (2) iterative—in which the same set of active sites is used repeatedly. Our study with PKS12 protein from Mtb (the largest protein in this genome) reveals a third mechanism for polyketide biosynthesis. In this hybrid "modularly iterative" mechanism, PKS12 protein forms a supramolecular assembly to perform repetitive cycles of iterations. The protein assembly is formed by specific intermolecular interactions between N- and C-terminus linkers, analogous to modular PKSs. Our study adds a new dimension to the existing catalytic and mechanistic versatility of PKSs, providing a new perspective on how metabolic diversity could be generated by different combinations of existing functional scaffolds.
Citation: Chopra T, Banerjee S, Gupta S, Yadav G, Anand S, Surolia A, et al. (2008) Novel Intermolecular Iterative Mechanism for Biosynthesis of Mycoketide Catalyzed by a Bimodular Polyketide Synthase. PLoS Biol 6(7): e163. https://doi.org/10.1371/journal.pbio.0060163
Academic Editor: Bradley Moore, University of California, San Diego, United States of America
Received: May 31, 2007; Accepted: May 27, 2008; Published: July 8, 2008
Copyright: © 2008 Chopra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: RSG is a HHMI International Research Scholar and is also supported by Swarnajayanti Fellowship from DST, India. This work was also supported by the Centre of Excellence grant from DBT, India to RSG and AS.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ACP, acyl carrier protein; AFM, atomic force microscopy; AT, acyl transferase; CD, circular dichroism; Cter, C-terminus linker; DH, dehydratase; ER, enoyl reductase; FAS, fatty acid synthase; HPLC, high performance liquid chromatography; KR, ketoreductase; KS, ketosynthase; MCoA, malonyl coenzyme A; MMCoA, methylmalonyl coenzyme A; Nter, N-terminus linker; PKS, polyketide synthase; PKS12Δ1, module 1-deleted polyketide synthase 12 protein; Sfp, surfactin phosphopantetheinyl transferase; TLC, thin layer chromatography
Polyketide synthases (PKSs) are multifunctional enzymes that assemble simple CoA-thioesters to form complex metabolites. These proteins are classified as Type I, Type II, and Type III based on their protein architecture. Type I PKSs contain various catalytic domains on a single polypeptide chain, which are functionally grouped to form a module. These are further classified as modular and iterative. In modular type I PKSs, each set of catalytic domains is used once during the entire cycle of biosynthesis, whereas iterative PKSs repeatedly use these functional domains [1–5]. Type II PKSs, on the other hand, assemble different catalytic domains to form a noncovalent multienzyme complex [6,7]. Both these types of PKSs require a carrier domain on which intermediates are tethered as acyl-thioesters. Type III PKSs are quite different and are classified along with other PKSs due to their mechanistic similarity in the process of 2-carbon condensations [8,9]. Type I and II PKSs utilize three core catalytic domains, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), to carry out decarboxylative condensations. The β-keto intermediates are processed by auxiliary domains; ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER), that dictate the overall chemistry of the metabolite.
Research in recent years has provided new themes that enhance the ability of PKSs to produce a wide array of metabolites that could mediate processes of significant functional importance. For example, AT-less Type I PKSs have been characterized, which utilize a discrete AT domain to load extender units in trans [10]. Fungal Type I iterative PKSs suggest new mechanistic basis of selective utilization of auxiliary reductase and methyl transferase domains [11–13]. The catalytic and mechanistic versatility of mycobacterial PKSs in generating complex virulent lipids have also been recently recognized [3,14]. A family of fatty-acyl AMP ligases has been demonstrated to facilitate biochemical crosstalk between fatty acid synthases (FASs) and PKSs [15]. An emerging family of type III PKSs involved in biosynthesis of novel resorcinolic lipids has also been characterized from Azotobacter vinelandii and Neurospora crassa [16,17]. Since metabolic diversity far exceeds our present understanding of the biosynthetic capability of this family of proteins, it is tempting to speculate that many new mechanistic and organizational features of PKSs remain to be decoded.
In this study, we report a novel mechanism of iterative catalysis for a bimodular multifunctional PKS12 (Rv2048c) protein from M. tuberculosis. This protein is involved in biosynthesis of a phosphoglycolipid called mannosyl-β-1-phosphomycoketide (MPM) [18]. The chemical structure of mycoketide lipid chain has been proposed to originate from repetitive condensations of methylmalonate and malonate units with medium-chain fatty acyl starters. Involvement of PKS12 protein in the biosynthesis of MPMs has been previously supported by genetic studies. By using a combination of biochemical, computational, mutagenic, analytical ultracentrifugation, and atomic force microscopy (AFM) studies, we propose that PKS12 protein is organized in a large supramolecular assembly mediated through specific interactions between the N- and C-terminus linkers (Nter and Cter, respectively). This assembly of PKS12 protein provides evidence for the intermolecular iterative mechanism for mycoketide biosynthesis. Our studies thus provide the first evidence for a novel "modularly iterative" mechanism for biosynthesis by PKSs.
Correspondence between Mycoketide Structure and PKS12 Catalytic Domains
The lipid portion of MPM, also referred to as mycoketide, consists of saturated alkyl chain with methyl branching at every fourth carbon (Figure 1). The total number of carbons could vary from C30 to C34 in different mycobacterial species [18]. Due to its similarity with mannosyl-β-1-phosphodolichol (MPD), initial structural characterization had suggested an isoprenoid mode of biosynthesis [19]. Based on genetic studies, PKS12 protein was recently shown to be involved in the biosynthesis of mycoketide. It is proposed that the biosynthesis would involve an iterative mode of catalysis. In mycobacteria, PKS12 is the largest protein, comprising 12 catalytic domains constituting two modules (Figure 1). Both these modules contain a complete set of catalytic and auxiliary domains that could add 2-carbon ketide units and also simultaneously carry out a three-step reduction to a saturated alkyl chain. Since mycoketide chemical structure contains branching at every alternate ketide unit, a possible iterative mechanism would involve alternate use of two modules wherein one module would condense a branched C3 ketide unit and the next would add a C2 unit. This would indicate that the two modules of PKS12 would be specific for methylmalonate and malonate extender units, respectively. The AT domains of PKSs characteristically show strict substrate specificity; however, there are examples in which the same AT domain could utilize both malonate and methylmalonate units [20]. Such promiscuity of AT domains in PKS12 could result in a complex mechanism of biosynthesis. We therefore decided first to dissect the specificity dictated by the two AT domains of PKS12 protein.
A schematic representation of bimodular PKS12 protein from M. tuberculosis, which is required for the biosynthesis of mycoketide. The two modules of PKS12 are colored in blue (AT specificity for methylmalonyl CoA) and buff (AT specificity for malonyl CoA), and the N- and Cters are indicated in green and red, respectively. Individual domains are drawn as small boxes in the two modules. Mycoketide chain originates from repetitive condensations of methylmalonate and malonate units with medium-chain fatty acyl starters. This saturated chain is released from PKS12 by an unknown mechanism, which is followed by reduction, phosphorylation, and mannosylation to form MPM.
Heterologous Expression of 431-kDa PKS12 Protein and Characterization of AT Domain Specificities
The 12,456-bp pks12 gene was cloned in T7 expression vector such that the protein would be expressed with a C-terminal His-tag. The N- and C- terminal ends were suitably engineered, and the central region of pks12 was spliced from overlapping shotgun library of mycobacterial D8 BAC clone. The phosphopantetheinylation of the ACP domain of PKS12 was achieved by coexpression of pRSG56 (a surfactin phosphopantetheinyl transferase [Sfp] expression plasmid) in BL21-DE3 strain of Escherichia coli [21,22]. Purification by Ni2+-NTA affinity chromatography followed by anion exchange chromatography showed a protein band running above the 212-kDa marker on the SDS-PAGE (Figure 2B, lane1). Since E. coli protein biosynthetic machinery is not programmed to synthesize such large proteins, and it is not easy to estimate exact molecular weight from SDS-PAGE for such large proteins, we performed peptide mapping by using mass spectrometry. Our analysis showed good coverage over the entire length of the protein, confirming expression of complete PKS12 protein. The specificity of AT domains was probed by using 14C malonyl coenzyme A (MCoA) and 14C methylmalonyl coenzyme A (MMCoA). SDS-PAGE gel-binding assays clearly showed that PKS12 could be radiolabeled with both of these substrates (Figure 2B, lanes 2 and 3). However, the specificity of individual AT domains to load extender units could not be determined. We therefore decided to engineer a single-module construct of PKS12.
(A) The PKS12Δ1 protein was constructed by deleting module 1 from the clone encoding PKS12 protein. The DNA encoding KS domains from module 1 and module 2 contains a conserved BsaBI restriction endonuclease site, which facilitated module 1 deletion. PKS12Δ1 retains N- and Cters of PKS12 protein. PKS12 AT2 null mutant (PKS12AT2°) and PKS12Δ1 AT null mutant (PKS12ΔAT°), both mutated at the catalytic Ser residue were constructed by using standard SDM protocols. The mutations in the domains are marked in red.
(B) Labeling of PKS12 protein with radioactive extender units. Lane 1 shows the Coomassie-stained, purified PKS12 protein (431 kDa). The protein could be labeled with both 14C-MCoA (lane 2) and 14C-MMCoA (lane 3). AT2 (S2672A) point mutant of PKS12 is labeled by 14C-MMCoA (lane 5) and not by 14C-MCoA (lane 4) depicting specificity of module 1 for MMCoA and module 2 for MCoA.
(C) Labeling of PKS12Δ1 protein with radioactive extender units. Lanes 1 and 2 show the Coomassie-stained, purified PKS12 (431 kDa) and PKS12Δ1 protein (234 kDa). PKS12Δ1 protein could be radiolabeled with 14C-MCoA (lane 3) but not with14C-MMCoA (lane 4). AT (S2672A) mutant protein of PKS12Δ1 does not incorporate any radioactive label (lanes 5 and 6).
Engineered Single-Module PKS12 Protein and Assignment of AT Domain Specificity
Based on previous understanding of engineering single-module PKS proteins, we utilized the in-frame BsaBI restriction sites present in the pks12 gene to delete module 1. This engineered PKS12Δ1 protein retained both the Nter and Cter and all the catalytic domains of module 2 (Figure 2A). PKS12Δ1 protein was expressed and purified analogous to PKS12 (Figure 2C, lane 2). The specificity of AT domain was again investigated by using radiolabeled substrates. PKS12Δ1 protein specifically utilized MCoA and no labeling of protein could be identified with MMCoA (Figure 2C, lanes 3 and 4). The specificity of this labeling was further confirmed by making a point mutation of the catalytic Ser amino acid residue of the AT domain. The S2672A null mutant protein, PKS12Δ1-AT°, was now incapable of loading any of these two extender units (Figure 2C, lanes 5 and 6). These studies clearly demonstrate specificity of module 2 for malonate unit and suggest that the module 1 of PKS12 protein would add a methylmalonate unit.
In order to unambiguously determine the specificity of AT1, we constructed an AT2 null mutant of PKS12, PKS12-AT2° (Figure 2A). This mutant protein could be specifically radiolabeled with 14C MMCoA and not with 14C MCoA (Figure 2B, lanes 4 and 5) thus demonstrating specificity of module 1 for MMCoA. These studies clearly demonstrate that the biosynthesis of mycoketide would involve alternate use of module 1 and module 2, respectively. The final assembly would involve five such cycles of iterations, and in terms of thiotemplate-based enzymology, would require transfer of growing polyketide chain from the ACP domain of module 2 to the KS domain of module 1. Such bimodular iterations during PKS biosynthesis have not been demonstrated. We therefore decided to investigate whether PKS12 protein by itself would be competent to produce mycoketide chain.
Cell-Free Reconstitution of PKS12 and PKS12Δ1 Protein
The catalytic activity of PKS12 protein was analyzed by incubating various starters (C6–C18) acyl-N-acetyl cysteamines (NACs) with radiolabeled extender units MCoA, MMCoA, and NADPH. Since PKS12 protein does not contain a chain-releasing domain, the enzyme-bound products were released by alkali hydrolysis. The extracted products were analyzed by performing radio-thin layer chromatography (TLC) experiments. Autoradiography analysis showed a diffused band at an Rf of about 0.5 (Figure3A). When either of the extender units was omitted from the assays, such broad bands were not observed (Figure S1). PKS12 protein showed preference for small-/medium-chain starter fatty acyl substrates. Radioactive bands observed at lower Rf were degradative products of acyl-CoAs and varied from assay to assay. Enzymatic assays of PKS12 protein with 9-hydroxy decanoyl NAC-thioester shifted Rf of the final product to about 0.2 (Figure3A, lane 8). Hydroxyl modification at ω-position of fatty acids dramatically alters the Rf value of products owing to the polar nature of starter unit and is an interesting approach to confirm enzymatic activity. Furthermore, incubation of PKS12 protein with KS inhibitor cerulenin showed no radioactive bands (Figure S2). We speculated that the diffused radioactive band could correspond to different intermediates formed during various stages of iterations. Our previous studies with another single module iterative enzyme, mycocerosic acid synthase, had shown presence of similar diffused bands [20]. The apparent catalytic turnover (kcat/KM) of the final product was estimated to be 42.4 μM−1 sec−1 (Figure 3B). This reconstitution provided confidence that PKS12 protein could indeed synthesize mycoketide metabolite. For experimental ease, detailed characterization of products was performed with PKS12Δ1 protein.
(A)Radio-TLC characterization of PKS12 products. Lane 1 to 8 show the propionyl, hexanoyl, octanoyl, decanoyl, lauroyl, palmitoyl, stearoyl, and 9-hydroxy decanoyl N-acetyl cysteamine thioester primed products. No product is formed in the absence of protein (lane 9).
(B) Time course of product formation catalyzed by PKS12 protein. The error bars correspond to three independent experiments, and the data points are an average of all three.
(C) Radio-TLC characterization of PKS12Δ1 products. Lane 1 to 8 show the propionyl, hexanoyl, octanoyl, decanoyl, lauroyl, palmitoyl, stearoyl, and 9-hydroxy decanoyl N-acetyl cysteamine thioester primed products. No product is formed in the absence of protein (lane 9).
(D) Time course of product formation catalyzed by PKS12Δ1 protein. The error bars correspond to three independent experiments, and the data points are an average of all three.
In order to biochemically reconstitute the PKS12Δ1 protein, we coincubated the protein with different acyl-NAC starters (C6–C18) along with NADPH and radiolabeled MCoA. Alkaline hydrolysis followed by radio-TLC analysis revealed substrate specificity for short-/medium-chain starters (Figure 3C). PKS12Δ1 showed robust activity with the apparent kcat/KM of 104.22 μM−1 sec−1 (Figure 3D). The activity was confirmed by constructing active site KS and AT null mutants, C2226A and the S2672A. Both these proteins were purified to homogeneity and were found to be catalytically inactive (Figure S3), thus confirming in vitro reconstitution of the PKS12Δ1 protein. A 2.5-fold improved catalytic activity of PKS12Δ1 along with better protein expression provided a good system to delineate the mechanisms involved in mycoketide formation.
In order to unequivocally characterize the PKS12Δ1 product, we designed a radio high performance liquid chromatography (HPLC)-based assay, wherein fatty acids with different carbon chain lengths (C3 to C18) were separated on a reverse-phase column by using a ternary solvent system. Products formed from the PKS12Δ1 reactions primed with hexanoyl-NAC starter unit were first analyzed on TLC plates. The diffused product band was eluted and further subjected to HPLC analysis. Six radioactive products could be detected in the standard assay (Figure 4A). PKS12Δ1 protein is expected to add two carbon units during each cycle of chain extension. As can be observed from Figure 4A, three of these products showed identical retention times with standard radioactive fatty acids (C12, C16, and C18) (standard spectra shifted to the left for clarity). Other radioactive peaks, based on retention times and on our understanding of the programming of PKS enzymology, would correspond to C8, C10, and C14, respectively. Radio-TLC analysis of these HPLC-purified products showed a systematic change in Rf (Figure 4B), which could be unambiguously confirmed with their standard fatty acids. Mass spectrometric analysis of these peaks further confirmed the identity of products. Particularly in the case of longer chain products, the isotopic 14C peak was enhanced as compared to the corresponding 13C peak (Figure 4C). The electrospray tandem mass spectrometry (ESI-MS/MS) pattern for 12C and 14C peaks showed expected dehydration and decarboxylation peaks (Figure 4D and 4E). Based on these studies, we propose that the single-module construct of PKS12 protein can perform up to six rounds of chain extension in our in vitro enzymatic assays.
(A) Characterization of products on HPLC. The radioactive product synthesized by PKS12Δ1 was analyzed on reverse-phase HPLC, and the elution times were determined by a radioactive detector. The reaction was primed with a hexanoyl starter that undergoes two carbon extensions with MCoA. The product peaks superimpose with radioactive standards. The traces are stacked with an x-axis offset for clarity.
(B) Autoradiograph of the HPLC-resolved PKS12Δ1 products that were concentrated and analyzed by radio-TLC. The Rf of various bands is C8 (0.468), C10 (0.476), C12 (0.492), C14 (0.507), C16 (0.523), and C18 (0.539).
(C) ESI-MS analysis of the 29.46-min HPLC peak. Molecular ion peak of 199.17 confirms the eluent to be lauric acid. Isotopic distribution is in concordance with radioactive substrates utilized in the assay.
(D) Tandem MS of [M-H]−1199.17 peak. The fragmentation pattern was similar to standard lauric acid corresponding to decarboxylation (154.93) and loss of water (181.17) from the parent ion.
(E) Tandem MS of the [M*-H]−1 14C peak of lauric acid 201.17 peak. The characteristic peaks show mass difference of 2, corresponding to 14C radioisotope.
Structural Analysis of PKS12 and N- and C-Terminus Linkers
Our in vitro characterizations of PKS12 protein validates that this bimodular protein could indeed synthesize mycoketide product. An intriguing feature of the PKS12 catalysis would be the long-range transfer of ketide chain from module 2 to module 1. The recent structures of FAS [23] and KS-AT didomain from erythromycin PKS [24] provide an opportunity to investigate this possibility in the three-dimensional space. PKSs are proposed to be X-shaped homodimeric proteins, in which KS, DH, and ER provide the majority of the interfacial contacts in a given module. Modules that are present on two separate polypeptide chains interact through small helices. These helices protrude out from the N- and C-terminus ends to form an end-to-end organization [2,25,26]. The mammalian FAS structure also indicates similar end-to-end packing for consecutive modules in two neighboring unit cells along the common two-fold axis of the dimer [23]. This organization is likely to be conserved for modules, which are present on a single polypeptide chain [2]. Figure 5A shows the model of PKS12 generated by docking two KS-AT-DH-ER-KR-ACP modules in an end-to-end orientation. The template for KS-AT-DH-ER-KR has been taken from mammalian FAS structure, whereas the relative orientation of ACP with respect to KS has been fixed based on the ACP-KS interactions as seen in the yeast FAS structure [27]. Based on our model, the distances between the catalytic centers of ACP2 and KS1 are in the range of 130 Å (Figure 5B). Despite the inherent flexibility of the ACP domain, chain transfer over such a large distance seems unfeasible. This then raises a paradoxical problem for the iterative biosynthesis of mycoketide by PKS12.
(A) The end-to-end orientation of PKS12 is shown. Module 1 of PKS12 is shown in cyan and module two in copper brown, except the KS and ACP domains. The KS domains are shown in green and the ACP domains in yellow.
(B) The active site residues of KS and ACP domains are shown in ball and stick, and the distances are shown.
(C) Model for the docking domains of PKS12 generated by SCRWL and energy minimized using CVFF force field of Insight II. The interacting residues of the N- and the Cters are shown.
Type I multifunctional PKSs synthesize polyketide products by either using an iterative mechanism with repetitive utilization of catalytic domains or by an assembly-line modular catalysis involving single use of active sites [28]. However, the programming that dictates these two mechanisms of biosynthesis has not been elucidated. Systematic analysis of various domains and linkers suggests an interesting comparison. The Cter modular proteins varies between 70–110 amino acids, whereas iterative linkers are typically smaller than 25 amino acids (G. Yadav and D. Mohanty; unpublished data). Moreover, a strong propensity to form helices could be observed in the case of modular linkers. A close examination of PKS12 protein suggested similarity to modular proteins, where the Cter is of 81 amino acids and also shows structural propensity to form three helices. This is in contrast to the Cters of two other mycobacterial iterative proteins, mycocerosic acid synthase (MAS) and PKS2 that have Cters of 11 residues and 13 residues, respectively, with no significant structural propensities. Furthermore, the analysis of the Nter of PKS12 also showed propensity to form a coiled-coil motif. Based on structural understanding of PKS proteins, the N- and C-termini cannot fold back to interact with one another. We therefore speculate that PKS12 protein molecules may form large supramolecular assemblies that interact with each other through the N- and C-terminus helices. We performed detailed analysis of PKS12 linkers to identify crucial residues involved in this docking complex. A chimeric model based on SCRWL [29] was built that utilizes the nuclear magnetic resonance (NMR) template of 6-deoxyerythronolide B synthase (DEBS) [25]. The residues in the helices of DEBS have been replaced by corresponding amino acids of PKS12, but the loops are retained (Figure 5C). The model was energy minimized using CVFF force field of Insight II [30]. Any two residues less than 5 Å apart were considered as interacting. This analysis led us to identify two key ionic interactions between C- and Nters (D4137 with H7 and R4147 with E20).
Analytical Ultracentrifugation and Mutagenic Analysis of Interacting Linker Amino Acid Residues
In order to explore macromolecular organization of PKS12, we resorted to sedimentation velocity studies using the analytical ultracentrifuge. This analysis provides a sensitive measure to ascertain whether the sample consists of single or multiple species [31]. At low salt concentrations, both PKS12 and PKS12Δ1 showed a spread in the sedimenting boundaries (Figure 6A and 6B). These data could not be fitted to determine a unique sedimentation coefficient and suggested a possibility of multiple stable aggregation states. At the same time, we also performed control experiments with other mycobacterial PKS proteins. Both PKS13 and PKS2, which are single-module proteins with molecular weight of 186 and 225 kDa, respectively, showed a single sedimenting boundary (Figure 6C and 6D). The spread observed for PKS12Δ1 was also investigated at different protein concentrations, and a similar spreading pattern was observed in the sedimentation velocity profiles. These studies clearly suggested that PKS12 protein forms a large supramolecular assembly, probably through intermolecular interactions mediated by N- and C-termini helices. PKS proteins are known to posses weak intermolecular interactions, as has been demonstrated for DEBS [32] and Pps proteins (O.A. Trivedi and R. S. Gokhale, unpublished data) by using gel filtration chromatography. The number of species observed in the analytical ultracentrifugation studies could thus correspond to different states of oligomerization. Similar sedimenting profiles of intermolecular association has been previously observed for histone proteins [33].
Analysis of PKS12 (A), PKS12Δ1 (B), PKS13 (C), and PKS2 (D) by sedimentation velocity in the XL1-analytical ultracentrifuge in 50 mM phosphate buffer (pH 7.2). The data were analyzed to yield the integral distribution of sedimentation coefficients.
Since intermolecular interactions in PKS12 protein is expected to be through the linker residues, we decided to completely swap the Nter residues with an N-terminus region from mycobacterial PpsA protein [20]. PpsA is the first module of the DIM biosynthetic machinery and contains an ACP loading domain at its N-terminus. This protein also does not show any coiled-coil region at its N-terminus end. This fusion construct of PKS12Δ1 showed extremely poor catalytic activity, thus emphasizing the significant role of these linker residues (Figure S3). We also performed analytical ultracentrifugation studies on this linker-change PKS12Δ1 (Figure S4). Interestingly, the scatter in sedimenting boundaries markedly observed for the wild-type protein was completely abolished. We then decided to carry out product analysis using radio-HPLC.
Analysis of the linker-change protein indicated that this protein could carry out only one round of malonyl-CoA condensation to synthesize C8 fatty acid (red trace in Figure 7). Clearly, this linker-change has abrogated the ability of PKS12Δ1 to perform iterations through formation of higher order oligomers. In order to directly explore the role of PKS12 linker residues, we decided to make site-specific mutants. Based on our PKS12 docking domain model (Figure 5C), we predicted a strong ionic interaction between E20 from N-terminus with C-terminus R4147 residue. The PKS12Δ1 E20S mutant protein showed a 5-fold decrease in its catalytic turnover. Quantitative radio-HPLC analysis also showed greater than 5-fold decrease in its product formation (blue trace in Figure 7). This compromised activity resulting from a change in one amino acid in its linker provides strong evidence of their requirement in efficient catalysis. H7D mutation remarkably led to enhancement of catalytic activity (black trace in Figure 7). Although the exact reason for this behavior is not clear, our computational analysis suggests that such a modification could provide favorable intrahelical contacts. Moreover, sequence alignment in this region is rather poor, and minor structural alterations can clearly provide a stabilizing effect. A double mutant constructed by combining these two mutations clearly showed an additive effect (green trace in Figure 7). The product profile was somewhat an average of two independent mutations. It is interesting to note that products formed from higher iterations were significantly lower in comparison to wild-type and H7D proteins. Clearly, single amino acid substitutions in the noncatalytic regions show effect on the catalytic rates of product formation, thus reemphasizing their importance in mycoketide biosynthesis through intermolecular iterative catalysis.
(A) The radioactive products synthesized by PKS12Δ1 and PKS12Δ1 mutant proteins were analyzed on reverse-phase HPLC, and the elution times were determined by using a radioactive detector. All reactions were primed by hexanoyl starter that undergoes two carbon extension(s) with MCoA. Trace for E20S mutant (blue) shows exact retention time while traces of all other mutant proteins are stacked with an x-axis offset for clarity; linker-change protein (red), H7D + E20S mutant (green), wild-type PKS12Δ1 (pink), and H7D mutant (black). Chain lengths of standard fatty acids eluting at these retention times are marked on these stacked traces. Value of 0.01 V is considered as background, based on the control experiments.
(B) Comparison of concentration dependent changes in the product profiles catalyzed by PKS12Δ1. Gradation in color indicates increase of protein concentration for each set of peaks, which in turn depict the product formed. At higher protein concentration, more of longer chain length products are synthesized, since there is a shift in equilibrium towards higher order multimeric species.
Protein Concentration-Dependent Changes in PKS12Δ1 Product Profile
Since intermolecular interactions show dependence on concentration, we examined the formation of products by PKS12Δ1 at different protein concentrations. For an intermolecular iterative catalysis, the formation of the final product would be dependent on the number of protein molecules participating in the multimeric assembly. We therefore hypothesized that formation of long-chain products would be facilitated at higher concentrations, whereas at lower concentration, the product profile should support biosynthesis of short-chain fatty acids. We investigated the product profile using radio-HPLC at three different concentrations of 0.97 μM, 1.94 μM, and 3.88 μM by using hexanoyl-NAC as the starter unit. As is evident from Figure 7B, the maximum amount of product formed at the lowest concentration corresponds to a single round of condensation of C2 unit. Moreover, all subsequent products are produced at lower amounts than the C8 product. At the highest protein concentration, there is favorable equilibrium towards formation of higher order multimeric species, and as can be observed from Figure 7B, there is a clear shift in product profile pattern, and C8 is the lowest of all the products. Such an effect would not be observed for proteins that would use an intramolecular iterative mode of catalysis.
Specific Inhibition of Catalysis by C-Terminus Linker Peptide
Since the supramolecular organization of PKS12 protein is proposed to be through N- and Cters, we wanted to investigate whether linker peptides would disrupt protein-protein interactions and thus affect catalysis. We therefore decided to assay PKS12Δ1 protein in presence of varying concentrations of purified Cter peptide. Towards this end, we cloned and expressed Cter peptide of PKS12 protein in BL-21 strain of E. coli by using T7 expression vector. Purification by Ni2+-NTA chromatography followed by anion exchange chromatography yielded PKS12 Cter peptide at high purity. Coincubation of this peptide with PKS12Δ1 protein showed a clear decrease in the formation of enzymatic products (Figure 8A, lanes 1–5). This was further confirmed and quantitated by radio-HPLC analysis of PKS12Δ1 reactions (Figure 8B), in which a marked change in product profile was observed. As a control, an equivalent concentration of bovine serum albumin protein was also used in the assay, which showed no effect on the enzymatic activity (Figure 8A, lane 7).
(A) Effect of Cter peptide, Cter-Nter concatenated peptide, and BSA on PKS12Δ1 catalysis. Specific inhibition of enzymatic activity by C-ter peptide can be seen from lanes 1 to 5. C-terminus peptide competes with other PKS12Δ1 molecules and thus specifically inhibits product formation. BSA and the concatenated C- and N-ter linker, which in itself forms a stable docking complex, does not interfere with PKS12Δ1 catalysis.
(B) Radio-HPLC analysis of PKS12Δ1 reaction inhibited by five times molar excess of Cter peptide. The solid curve indicates the control run without any Cter peptide, and the dotted curve shows the profile for inhibited assay.
(C) AFM analysis of PKS12Δ1 showing multimeric organization.
The specificity of inhibition by Cter peptide was further investigated by using a covalently concatenated Cter–Nter peptide of PKS12. Such a Cter-Nter fusion construct was previously used for structural elucidation of docking domain in erythromycin polyketide synthases, in which these linkers form a stable docking complex by forming a pseudo four-helix bundle [25]. The Cter-Nter peptide was expressed in E. coli by splicing Nter at the end of the Cter sequence. The propensity of Cter and Cter-Nter peptides to adopt helical conformations was investigated by using circular dichroism (CD). The ratio of the negative molar ellipticities at 220 and 208 nm is 0.65 for Cter peptide and 1.08 for Cter-Nter peptide. Ratio of approximately 1.0 has been suggested to be typical of interacting α-helices [34,35]. Taken together, these CD studies suggest that Cter peptide forms α-helices, which together with Nter peptide forms an interacting pseudo four-helix bundle. Enzymatic assays performed in the presence of Cter-Nter peptide showed no inhibition of PKS12Δ1 catalytic function (Figure 8A, lane 6). These studies thus demonstrate that Cter peptide specifically perturbs PKS12Δ1 oligomerization by competitively interacting with the Nter region of PKS12Δ1 protein.
Atomic Force Microscopy Analysis of PKS12Δ1
Multimeric organization of PKS12 protein was further validated by performing atomic force microscopy studies. PKS12Δ1 protein from the assay mixture was used for visualization under noncontact acoustic AC mode. Figure 8C shows the presence of singular particles and some linear oligomers randomly distributed over the entire surface. Singular particle dimensions as analyzed by SPIP software could correspond to native monomers of PKS12Δ1. Interestingly, a distinctive pattern of linear multimeric species of varying length could also be observed. Using the height scaling method for these measurements, the linear multimeric species are not loosely interacting groups of single particles, instead, are professedly to be unified entities. These studies reinforce association between protein molecules.
Intermolecular Acyl Chain Transfer between Two Proteins
The unambiguous proof of intermolecular iterative mechanism of biosynthesis for mycoketide was established by designing an assay in which direct transfer of acyl chain from ACP of one protein molecule onto the KS domain of another PKS protein molecule could be demonstrated. Towards this end, we cloned and purified the ACP2 domain along with the Cter (ACP2Cter) of PKS12. The ACP2Cter protein was expressed in the apo-form and then Sfp protein was used to directly load 14C-hexanoyl phosphopantetheine group onto the catalytic Ser of ACP [36]. Coincubation of this protein with PKS12Δ1 showed facile transfer of radioactivity to this large PKS protein (Figure 9, lane 1). This transfer was not observed for the KS active site mutant protein of PKS12Δ1 (C2226A) (Figure 9, lane 2). The specificity of the interactions was further investigated by using another ACP protein (Rv1344) from M. tuberculosis. Although the Rv1344 protein could be acylated with 14C-hexanoyl phosphopantetheine group, no transfer could be seen on incubation with PKS12Δ1 (Figure 9, lane 3). Appropriate control experiments showed no direct labeling of PKS12Δ1, both in the presence or absence of Sfp protein (Figure 9, lanes 4 and 5). Taken together, these studies explicitly demonstrate specificity of intermolecular interactions and also of transfer of acyl chain from the phosphopantetheine group of ACP to the catalytic Cys residue of the KS domain.
Sfp from B. subtilis was used to label ACP2Cter of PKS12 (lane 1 and 2) and a noncognate ACP protein, Rv1344, from M. tuberculosis with 14C hexanoyl CoA. Although ACP2Cter was able to transfer the label to PKS12Δ1 (lane 1), no transfer was seen when either the KS mutant of PKS12Δ1 was used (lane 2) or when a noncognate ACP with PKS12Δ1 was used (lane 3). Minus ACP control experiment did not show any label on PKS12 (lane 4); there was also no direct labeling of PKS12Δ1 observed (lane 5)
This study unravels a novel biosynthetic mechanism by which mycobacterial PKS12 protein is involved in the biosynthesis of the acyl chain of MPMs. Cell-free reconstitution of a 431-kDa PKS12 protein along with systematic biochemical analysis has facilitated characterization of a unique supramolecular organization. We show that this assembly is mediated through precise intermolecular interactions between its N- and Cters in a head-to-tail association. The organization of this supramolecular assembly could be explained based on the recent three-dimensional structures of FASs [23] and the high resolution structure of KS-AT didomain from module 5 of erythromycin PKS [24]. The overall X-shaped dimeric structure of FAS is built around KS dimer interface. DH and ER domains also provide stability to this homodimeric protein, whereas AT and KR catalytic domains are located away from the dimer interface. It is clear from these structures that the ACP domain undergoes large conformational changes. The phosphopantetheine arm on ACP is expected to reach out to various domains within the same module and also to the KS domain of the next module to facilitate catalysis in these modular proteins [26].
The noncovalently associated cognate modules have been proposed to interact through C- and N-terminus helices that form an independent docking complex [21,25]. As demonstrated in the DEBS KS-AT structure, the N-terminus helix protrudes away from the KS dimer interface and forms a stable four-helix bundle with C-terminus helices of the previous module. The two homodimeric modules thus are arranged in an end-to-end fashion [26]. Since positions of modules could be changed by suitable engineering of linkers, the organization of two covalently linked contiguous modules is expected to follow similar organization [2,21]. PKS12 protein, investigated in this study, possesses a bimodular organization, which was proposed earlier to catalyze five rounds of alternative condensations of methylmalonyl and malonyl units through an iterative mechanism. Such a biosynthetic route would require transfer of intermediate chain from ACP of module 2 to KS of module 1. Based on present understanding of modular assembly, this chain transfer seems unfeasible. Therefore, in this study, we have systematically dissected programming of PKS12 to biosynthesize mycoketide.
In recent years, "stuttering" as well as "skipping" of modules has been reported during biosynthesis of polyketides that deviate from the linear processive mechanistic logic of modular biosynthesis [37–39]. The modular PKSs of borrelidin and aureothin are two recent examples that illustrate a controlled iterative utilization of selective modules during polyketide biosynthesis [40,41]. We therefore first confirmed the specificity of AT domains from two modules of PKS12 to confirm that the two modules are used alternately during mycoketide biosynthesis. This experimental confirmation is in agreement with our previous studies, in which we had demonstrated that a crucial Phe residue acts like a gatekeeper that discriminates between malonate and methyl malonate specificity [42]. The module 2 of PKS12 protein indeed possesses this important Phe residue that restricts its specificity to malonyl extender unit. The corresponding amino acid in module 1 is Ser and therefore is able to utilize methylmalonyl extender units.
In vitro enzymatic assays for PKS12 and PKS12Δ1 confirmed that this protein could indeed synthesize mycoketide. Detailed analysis of the N-ter and C-ter sequences suggested a modular mode of catalysis for PKS12. The organization of PKS12 proteins to form a larger molecular assembly was demonstrated by analytical ultracentrifugation. The significance of this organization was investigated by replacing N-ter and also by site-specific mutagenesis of crucial amino acids. Although the linker-change protein led to a single round of chain extension, the single amino acid mutants showed appreciable change in overall catalysis. Similar studies earlier with DEBS protein has shown crucial role of single amino acids in forming a docking complex [43]. More evidence for intermolecular iterative catalysis was provided by investigating the concentration-dependent changes in the product profiles of PKS12Δ1. At higher protein concentration, more of longer chain products are synthesized, since there is a shift in equilibrium towards higher order multimeric species. Furthermore, the Cter showed specific inhibition of enzymatic activity by disrupting protein–protein interactions while competing for the cognate N-ter of PKS12 protein. Quite remarkably, the docking domain consisting of Cter-Nter concatenated protein does not inhibit catalysis. CD analysis of these linker peptides supports the formation of docking domains through interacting helices. The imaging studies of PKS12Δ1 performed by using AFM also clearly showed propensity to form oligomeric structures. An unambiguous proof of this novel organization comes from our experiment in which we demonstrate specific transfer of acyl chain from (n − 1) protein to the next protein (n), which are designed to specifically interact through C- and Nters, respectively.
PKS12 protein thus forms a modular assembly to perform repetitive condensation analogous to iterative proteins. We therefore refer this mechanism of catalysis as "modularly iterative" (Figure 10). To the best of our knowledge, this is probably the first example wherein intermolecular iterative catalysis has been characterized by forming a novel structural organization. Interestingly, two recent studies have provided evidence for the formation of a large, singular mega-complex involving several PKS and FAS proteins [44,45].
Supramolecular organization of PKS12 protein formed by interactions between N- and Cters.
Biosynthesis of MPMs would require other downstream enzymes that would release and phosphorylate the nascent acyl chain and then carry out final mannosylation. The computational analysis of this region in the Mtb genome suggests few putative enzymes that could be involved in the complete biosynthesis of MPMs. Associated with this cluster is LipT, possessing an esterase motif, which could release the mycoketide from PKS12, similar to the lovD protein from lovastatin biosynthesis [11]. Alternatively, Rv2047c could reductively release alkyl chain from PKS12 protein. Rv2047c protein shows homology with recently characterized reductive domains from multifunctional non-ribosomal peptide synthetases. This protein also contains a swivel domain commonly found in phosphoenol pyruvate (PEP)-utilizing enzymes that may be required for phosphorylation. Located upstream of PKS12 is Ppm1, encoding a polyprenol monophosphomannose synthase that could mannosylate the phophorylated alkyl chain [46].
The novel intermolecular iterative biosynthetic mechanism identified in this study provides new perspective to our understanding of polyketide biosynthetic machinery. This study also highlights clever ways by which mycobacteria utilize polyketide biosynthetic machinery to produce exotic lipid metabolites.
Materials and Methods
The bacterial artificial chromosome (BAC) library of M. tuberculosis H37Rv was obtained from the Pasteur Research Institute, France. Radioactive malonyl CoA were purchased from Perkin Elmer Life Sciences (1.9166 GBq/mmol) and radioactive methylmalonyl CoA was purchased from American Radiolabeled Chemicals (55 mCi/mmol). E. coli XL1-blue and E. coli BL21 (DE3) were used as cloning and expression strains, respectively. All other chemicals used were of analytical grade.
Cloning of pks12 and and pks12 constructs.
BAC genomic DNA of M. tuberculosis H37Rv [47] was digested with restriction enzyme NheI, and the fragments were cloned in a NheI-digested vector, pTC1. Cloning vector pTC1 has NdeI to HindIII multiple cloning site region of pET28c inserted in the EcoRV site of pBluescript-SK+ (pBS) vector. NheI site was specifically utilized to obtain a 14,412-bp genomic DNA fragment, which includes the complete pks12 gene. Gene pks12 was cloned in two parts from pTC2 which contains this 14,412-bp fragment. In brief, the N-terminus 827 bp of pks12 was amplified (primers RSG675 to RSG678) by overlap extension PCR while inactivating an NdeI (632 bp) site via silent mutations to facilitate cloning. This NdeI (1 bp) to PstI (827 bp) fragment was cloned in the SnaBI site of pLITMUS 28, and the construct was further used to insert a PstI (827bp)–PstI (6,896 bp) fragment from pTC2 to yield pTC5. The C-terminus 1,379-bp fragment was PCR amplified using primers RSG679 and RSG681. The AscI (11,077 bp)–HindIII (12,456 bp) fragment was cloned in pNEB193 followed by insertion of NdeI (6,701 bp) to AscI (11,077 bp) fragment from pTC2. This NdeI (6,701 bp) to HindIII (12,456 bp) fragment was inserted in pET21c (+) followed by insertion of NdeI (1 bp)–NdeI (6,701 bp) from pTC5 to create PTC10, the expression plasmid for PKS12 with a C-terminal polyhistidine tag. Primer sequences used for cloning are given in Table S2.
pks12Δ1 was constructed by deleting BsaBI (112 bp)–BsaBI (6,178 bp) fragment from pTC5. The clone thus obtained was called pTC13. pTC13 was utilized to insert the NdeI (1 bp) to NdeI (6,701 bp) fragment containing this deletion alongside NdeI (6,701 bp) to HindIII (12,456 bp) fragment in pET21c (+). The clone obtained was called pTC15 and was used for the purification of PKS12Δ1.
The linker-change mutant of pks12 was constructed by replacing the N-terminal NdeI to BsaBI fragment of pks12 with the N-terminal NdeI to BsaBI fragment of ppsA gene by using the conserved BsaBI site present at the start of the KS domain. Although BsaBI site was already present in pks12, it was engineered in ppsA via silent mutations.
For construction of the PKS12 Cter peptide, the C-terminus 243 bp was PCR amplified (primers RSG1505, RSG1506) from pTC2 while engineering restriction sites at the terminal ends to facilitate cloning. For concatenated Cter-Nter fusion construct, Cter and Nter were separately PCR amplified (primers RSG1426, RSG1427 for Cter and RSG1428, RSG1430 for Nter) while engineering a PstI site at the end of Cter and PstI site at the beginning of Nter to facilitate their joining.
AT2 null mutant of PKS12 was constructed by insertion of NdeI (1 bp)–NdeI (6,701 bp) from pTC5 in NdeI-digested AT mutant of PKS12Δ1, pTC23.
ACP2Cter was PCR amplified (primers RSG 1718, RSG 681) and cloned in pET21c (+) expression vector.
Site-directed mutagenesis.
Site-directed mutagenesis was used to generate the point mutants of pks12Δ1. The mutations are shown in Table S1, and the mutagenic primers are shown in Table S2. To minimize errors, small fragments containing the region of interest were subcloned and inserted back after mutations were introduced. All mutants were constructed by using Quickchange Site Directed Mutagenesis Kit (Stratagene). Mutations were confirmed by restriction analysis and by DNA sequencing.
Expression and purification of proteins.
All proteins were expressed and purified using the following procedure. The expression plasmids were cotransformed in BL21-DE3 strain of E .coli with pRSG56 to enable coexpression of Sfp for phosphopantetheinylation of the ACP domain wherever required. The cells were grown at 30 °C in LB medium with 50 μg ml−1 of antibiotic (carbenicillin/kanamycin) to an absorbance of 0.6 at 600 nm. The cells were incubated on ice for 10–15 min, and induced with 0.1 mM to 1 mM isopropyl thio–β-D-galactoside for 12–18 h at an appropriate temperature (22 °C to 37 °C). The cells were harvested by centrifugation (3,500 g, 10 min; Beckman Coulter Allegra 6R), resuspended in buffer A (50 mM Tris chloride [pH 8.0] containing 10% glycerol) containing 150 mM NaCl, lysed using a French press, and cellular debris was removed by centrifugation (24,000 rpm, 40 min; Sorvall evolution RC, rotor: SA 300). Polyethylene imine was added to a final percentage of 0.1%, and DNA pellet was removed by centrifugation (20,000 rpm, 20 min; Sorvall evolution RC, rotor: SA 300). Ni2+-nitrilotriacetic agarose resin was added to the supernatant (0.75 ml l−1 of culture), and the proteins were purified using affinity chromatography with increasing concentration of imidazole in buffer A. Anion exchange chromatography was performed by using a 6-ml Resource-Q column and AKTA chromatography system (Amersham). The protein was purified in buffer B (100 mM phosphate buffer [pH 7.2] containing 10% glycerol) and eluted with an increasing gradient of 1 M NaCl in buffer B with a flow rate of 2 ml min−1.
ACP2Cter expressed as inclusion bodies at all temperature and IPTG concentrations tested. After French press, the pellet containing inclusion bodies was resuspended in buffer containing 2 M Urea in 100 mM phosphate buffer (pH 7.2), 10 mM Tris buffer (pH 8.0) and 1 mM DTT. Supernatant was separated from the 2 M pellet by centrifugation (15,000 rpm, 30 min), the pellet was resuspended in a higher concentration of urea until the concentration reached 8 M, and supernatant was separated at each stage. SDS-PAGE analysis of all the fractions showed presence of ACP2Cter in 2 M fractions. This suspension was subjected to a 100-fold "snap-dilution" in the refolding buffer containing 50 mM Tris, 10% Glycerol and 150 mM NaCl. The refolded protein was purified and used for transfer assays.
Protein concentrations were determined by using BCA protein estimation kit (Pierce).
Labeling of enzymes by 14C-methylmalonyl- and 14C-malonyl-CoA.
Enzymes (1–5 μM) were incubated with 14C-MCoA and 14C-MMCoA (36 μM) at 4 °C for 2 min. The reactions were quenched by addition of SDS-PAGE loading dye lacking any reducing agents like dithiothreitol or β-mercaptoethanol. Samples were directly loaded on a 6% SDS-PAGE gel, and electrophoresis was performed at 25 mA until the dye front ran out. The gel was dried and analyzed using a phosphorImager (Fuji BAS5001).
Product formation assays.
PKS12 enzymatic assays included 100 mM phosphate buffer (pH 7.2), 150 μM acyl-NACs, 75 μM MCoA, 75 μM MMCoA, 7.2 μM 14C-MCoA, 7.2 μM 14C-MMCoA, 4 mM NADPH, 10% glycerol, 2 mM Tris (2-carboxy ethyl) phosphine hydrochloride (TCEP), and 2.5–4.5 μM protein in 100-μl reaction volume. The reactions were incubated at 30 °C for 6–12 h. The products were released with 45% KOH followed by acidification and extraction in hexanes or ethyl acetate. The extract was spotted on TLC plate that was developed by using 40:60:1 hexanes:ether:acetic acid. The radio-TLC plates were analyzed by using a phosphorimager. For PKS12Δ1, the enzymatic assays included all the components except MMCoA. For radio-HPLC assays, the concentration of 14C-MCoA was increased to 25 μM while keeping the total MCoA concentration as 100 μM. The band of interest was scraped from TLC, extracted in ethyl acetate, concentrated and injected on C18 reverse-phase column (gradient: 100% B in 20 min, 100% B in 40 min, 20% B in 60 min; A: H2O, B: 5% methanol in ACN with 0.1% formic acid) on HPLC (Shimadzu). To analyze the HPLC eluted peaks, PKS12Δ1 products were first run on TLC plates, these were then extracted and separated on HPLC and then further analyzed on TLC plates. The radioactivity was monitored online by a radioactive detector (IN/US system β-RAM Model 3). For kinetic analysis, radiolabeled products were quantified using PhosphorImager (Fuji BAS5001).
For competition experiments, the competing proteins (Cter, Cter-Nter, and BSA) were titrated into reaction mixtures containing 100 mM phosphate buffer (pH 7.2), 75 μM MCoA, 7.2 μM 14C-MCoA, 4 mM NADPH, 10% glycerol, 2 mM TCEP, and 2.5 μM protein in 100-μl reaction volume. The reactions were analyzed in a manner similar to PKS12Δ1 assays.
ACP to PKS transfer assays.
The ACP to PKS transfer assays were carried out in two steps wherein first the ACP protein was exogenously labeled with 14C Hexanoyl CoA in a reaction containing 45 μM 14C Hexanoyl CoA, 12.5 mM MgCl2, and the Bacillus subtilis enzyme Sfp [48]. The reaction was carried out for 15 min at 37 °C, quenched with 0.05 M EDTA, and PKS proteins were added to a final concentration of 1.5 to 2 μM. The reaction mixture was incubated for 15 min at 30 °C for the transfer to occur. The reactions were quenched by addition of SDS-PAGE loading dye lacking any reducing agents, such as dithiothreitol or β-mercaptoethanol. Samples were directly loaded onto a 10% SDS-PAGE gel, and electrophoresis was performed at 25 mA. The gel was dried and analyzed using a Phosphorimager (Fuji BAS5001).
Synthesis of alkyl NAC thioesters.
Various alkyl NAC thioesters and 9-hydroxy decanoyl NAC was synthesized according to a modified protocol reported earlier [20,49]
Sequence analysis and structural modeling.
Primary sequence of the gene was obtained from Tuberculist (http://genolist.pasteur.fr/TubercuList/). Secondary structure analysis and multiple sequence alignment for the PKS12 chimera model were carried out by using seqweb software. Domain boundaries for PKS12 were obtained from SEARCHPKS, a program developed for detection and analysis of PKS domains (http://rd.plos.org/pbio.0060163). The chimera model for linkers was built using SCRWL [29] in which the intervening loops of the DEBS docking domains were kept and only the interacting helices were changed to identify putative interacting residues. The model for bimodular PKS12 was generated by docking KS-AT segment of module 2 with DH-ER-KR segment of module1 using the software 3D-DOCK [50]
Sedimentation velocity analysis.
Sedimentation velocity experiments were conducted with an optima XLI instrument (Beckman Coulter) using an An 60Ti four-hole rotor at 35,000 rpm at 20 °C with three 420-μl samples in standard double-sector Epon centerpieces equipped with quartz windows. The sample A280 ranged from 0.6–0.9. Sedimentation velocity data were collected using continuous scan mode set at 0.001-cm radial increments. The scans were analyzed by the method of Van Holde-Weischet [51]. These data yielded the integral distribution of sedimentation coefficients plotted as the boundary fraction versus S20,w (sedimentation coefficient corrected for water at 20 °C )
Atomic force microscopy.
Pico plus atomic force microscope (Agilent Technologies) was used in acoustic AC (noncontact) mode for imaging. Images were recorded in air with either a bare mica surface or mica with the samples (normal spring constant of cantilever: 2.8 N/m, frequency: 59.722 kHz). The PKS12Δ1 reaction mixture was diluted 20-fold with 100 mM phosphate buffer and was immobilized on freshly cleaved mica for 30 s. The sample was washed with nanopure water, dried under N2, and subjected to AFM analysis.
Circular dichroism analysis.
CD spectra were recorded on a Jasco-710 instrument equipped with a temperature cell holder. The spectra were an average of ten scans, and the data were presented as mean residual ellipticity (MRE) expressed in deg cm2 dmol−1. The protein concentrations were determined by using BCA protein estimation kit (Pierce).
Supporting Information
Figure S1. PKS12 Activity in Absence of Any of the Two Extenders
PKS12 protein was assayed for activity as given in Materials and Methods. While PKS12 was active in presence of both extenders (lane 1), absence of MCoA (lane 3), or MMCoA (lane 2) from the assays rendered the protein inactive.
Figure S2. Effect of KS Inhibitor, Cerulenin, and Heat Inactivation on Enzymatic Assays of PKS12
PKS12 protein (lane 1), PKS12 protein with 100 μM cerulenin (lane 2), and PKS12 protein with ethanol equivalent to the volume of cerulenin added (lane 3) were incubated at 30 °C for 1 h prior to setting up assays as given in Materials and Methods. Extractions were done in hexanes to extract only the polyketide product. Cerulenin renders the KS domain inactive by forming a covalent adduct and inhibits the reaction (lane 2). Preincubation (lane 1) and ethanol (lane 3) do not lead to any inhibition. PKS12 protein activity was further confirmed by denaturing the protein by boiling at 100 °C for 10 min and setting up assays (lane 4).
Figure S3. PKS12Δ1, KS Mutant, AT Mutant, and Linker-Change Protein Activity Assays
PKS12Δ1, KS mutant (C2226A), AT mutant (S2672A), and linker-change protein were assayed for activity as given in Materials and Methods. Although PKS12Δ1 showed robust activity (lane 1), KS mutant (lane 3) and AT mutant (lane 4) are totally inactive. Lane 5 shows the activity assays for the linker-change protein, and no background activity is seen in the absence of protein (lane 2).
Figure S4. Analytical Ultracentrifugation Analysis of Linker-Change Mutant
Analysis of linker-change mutant of PKS12Δ1 by sedimentation velocity in the XL1-analytical ultracentrifuge in 50 mM phosphate buffer (pH 7.2). The data were analyzed to yield the integral distribution of sedimentation coefficients.
Table S1. Highlighting Mutations for Site-Directed Mutagenesis of Key Residues
Mutations are indicated in bold
Table S2. List of Primers Used
Author Contributions
TC carried out cloning, expression, purification, and biochemical characterization of pks12 and all other related constructs. SB carried out analytical ultracentrifugation studies in RPR's laboratory. TC and GY carried out the bioinformatic analysis of pks12. SA built the PKS12 model in DM's laboratory. SG and AS contributed to AFM studies. RSG directed the project. TC, DM, and RSG compiled results and wrote the manuscript.
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- 39. Wenzel SC, Kunze B, Hofle G, Silakowski B, Scharfe M, et al. (2005) Structure and biosynthesis of myxochromides S1–3 in Stigmatella aurantiaca: evidence for an iterative bacterial type I polyketide synthase and for module skipping in nonribosomal peptide biosynthesis. Chembiochem 6: 375–385.
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Olano C, Wilkinson B, Moss SJ, Brana AF, Mendez C, et al. (2003) Evidence from engineered gene fusions for the repeated use of a module in a modular polyketide synthase. Chem Commun (Camb) Nov. pp. 2780–2782.
- 42. Yadav G, Gokhale RS, Mohanty D (2003) Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases. J Mol Biol 328: 335–363.
- 43. Weissman KJ (2006) Single amino acid substitutions alter the efficiency of docking in modular polyketide biosynthesis. Chembiochem 7: 1334–1342.
- 44. Straight PD, Fischbach MA, Walsh CT, Rudner DZ, Kolter R (2007) A singular enzymatic megacomplex from Bacillus subtilis. Proc Natl Acad Sci U S A 104: 305–310.
- 45. Veyron-Churlet R, Guerrini O, Mourey L, Daffe M, Zerbib D (2004) Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability. Mol Microbiol 54: 1161–1172.
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- 48. Cox RJ, Crosby J, Daltrop O, Glod F, Jarzabek ME, et al. (2002) Streptomyces coelicolor phosphopantetheinyl transferase: a promiscuous activator of polyketide and fatty acid synthase acyl carrier proteins. J Chem Soc Perkin Trans 1: 1644–1649.
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Introduction to Modeling Surface Reactions in COMSOL Multiphysics®
Edmund Dickinson July 13, 2017
In biophysics, electrochemistry, and the design of catalytic reactors, researchers and engineers exploit the special chemical and physical properties of solid surfaces involving both gas-solid and liquid-solid interfaces. This blog post discusses the basics of the kinetics of surface reactions at simple surfaces and how they can be modeled with the COMSOL Multiphysics® software. In a subsequent blog post, we will look at how mass transport and reaction kinetics at surfaces are described for homogenized porous media.Read More
Claire Bost June 14, 2017
When ambient air flows through porous media, it carries moisture. In this process, temperature and moisture are coupled: The vapor saturates depending on the temperature conditions, while latent heat effects due to evaporation and condensation modify the temperature. We discussed heat and moisture transport in air in a previous blog post. Let's address the specific transport processes we need to consider in pores and how to model heat and moisture transport in porous media with the COMSOL Multiphysics® software.Read More
Bridget Cunningham April 26, 2017
Sometimes when you bake a cake, it doesn't turn out how you expected. Part of this is due to the underlying heat and mass transfer phenomena that occur within the baking process, which affect the end result. With tools like the COMSOL Multiphysics® software, you can study and predict how these mechanisms work and use this knowledge to bake a better cake.Read More
Edmund Dickinson March 14, 2017
In corrosion analysis, we often want to study corroding surfaces whose electrical connectivity is not as simple as a controlled current or voltage. Instead, an electrode surface might be short circuited to another electrode through a direct connection, such as the electrical connection between a monopile and transition piece. Here, we look at how to describe these electrodes and external short circuits using the appropriate boundary conditions in the COMSOL Multiphysics® software.Read More
Bridget Cunningham March 10, 2017
While reverse electrodialysis (RED) is a promising source of renewable energy, it can be a challenging process to analyze. The performance of RED units is affected by the physical phenomena that occur when converting salinity gradient energy into electric current. To address this, one team of researchers used a novel approach to model such systems in the COMSOL Multiphysics® software. Their multiphysics model and subsequent simulation studies provide further insight into designing and optimizing RED units.Read More
Caty Fairclough March 6, 2017
One way to design utility boilers with high thermal efficiency is to improve their furnaces — the most important part in their energy conversion process. Studying furnaces in utility boilers requires engineers to account for radiation, which can be difficult to solve for analytically and expensive to study experimentally. As an alternative, we can use the COMSOL Multiphysics® software to analyze radiative heat transfer in utility boiler furnaces and improve their designs.Read More
Edmund Dickinson February 9, 2017
Electrochemical impedance spectroscopy is a versatile experimental technique that provides information about an electrochemical cell's different physical and chemical phenomena. By modeling the physical processes involved, we can constructively interpret the experiment's results and assess the magnitudes of the physical quantities controlling the cell. We can then turn this model into an app, making electrochemical modeling accessible to more researchers and engineers. Here, we will look at three different ways of analyzing EIS: experiment, model, and simulation app.Read More
Ed Fontes February 2, 2017
Fat-washing cocktails has become popular in the last decade. This technique has made it possible for bartenders to create drinks such as the Benton's Old-Fashioned, a bacon-infused bourbon cocktail, and a milder pecan-butter-infused bourbon. In this blog post, we discuss this innovative cocktail technique and how it can be transferred to an industrial scale. In fact, many chemical processes in a variety of industries already use similar methods.Read More
Bridget Cunningham January 13, 2017
For automotive designers, developing effective evaporative emission control systems in vehicles is an important task. Without these systems, volatile hydrocarbons can escape from a vehicle's fuel tank, producing air pollution and smog. The COMSOL Multiphysics® software provides the features and functionality needed to model these systems in order to understand their operations and improve their performance. Here's a look at one research team's analysis of an evaporative emission control system for bioethanol-blend fuels.Read More
Caty Fairclough December 15, 2016
For patients with renal failure, efficient dialysis treatment is vital. One point of focus is designing high-performance dialysis equipment that increases contaminant removal, improving treatments like hemodialysis. To accomplish this, you can study aspects of the hemodialysis process, such as membrane dialysis devices, with numerical modeling apps. These apps, like the one discussed here, enable users to more quickly analyze the effects of different inputs and improve designs.Read More
Bridget Cunningham December 7, 2016
Traditional lithium-ion batteries use an electrolyte based on a flammable liquid solvent, which can cause them to catch fire if they overheat. In recent years, nonflammable solid electrolytes have been investigated as an alternative to improve battery design and safety. Optimizing this technology for industrial applications, however, requires a better understanding of the electrochemical processes inside the device. Simulation serves as a valuable tool for this purpose, helping to realize the use of solid-state lithium-ion batteries in the near future.Read More

subset: commoncrawl


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Two brothers whose Rosedale auto body shop became the focus of a federal investigation into widespread corruption in the Baltimore Police Department were sentenced to prison Wednesday.
Hernan Alexis Moreno, 32, of Rosedale and Edwin Javier Mejia, 29, of Middle River received prison terms of 33 months and two years, respectively, for paying officers to bring them business. The kickbacks scheme was uncovered last year.
Their sentences conclude a federal case that sullied the reputation of the Police Department, implicating roughly 60 officers and resulting in 15 being sentenced in federal court to prison terms between eight and 42 months. Another officer pleaded guilty to theft in state court.
Rod J. Rosenstein, the U.S. attorney for Maryland who prosecuted the cases, called the FBI's investigation of the Majestic Auto Repair scheme "as professional an investigation of police corruption as we've ever seen." He credited an FBI wiretap that caught many of the participants discussing the scheme on tape with bringing about guilty pleas fairly quickly.
"It was really the wiretap that resulted in the discovery of all these corrupt police officers and produced irrefutable evidence of their part in the crimes," Rosenstein said.
Anthony Guglielmi, a Baltimore police spokesman, said Wednesday that the closure of the case is "bittersweet" in that it is disappointing officers broke the law but gratifying that the department's own identification of the problem and collaboration with the FBI and Rosenstein brought it to an end.
Acting Commissioner Anthony Barksdale has said previously that he is looking forward to closing "an embarrassing chapter for the department" and is glad the officers involved "were held accountable for their actions."
The officers were found guilty of taking cash payments from Moreno and Mejia in return for referring motorists involved in accidents to Majestic, which opened in 2006 and was not authorized to do business with the city.
Some officers also took their own cars to Majestic for fraudulent claims, falsified police reports and ignored Majestic's effort to increase vehicle damage to boost insurance payouts, according to court records.
According to the brothers' plea agreements, they first arranged in 2008 to pay hundreds of dollars to two city officers, Jhonn Corona and Rodney Cintron, in exchange for the officers' directing business their way.
Moreno testified in federal court that he paid $300 per referral to more than 60 officers, and his business grew so quickly that he had to expand and manage a backlog of customers.
Rosenstein said he hopes the case will motivate officers to report misdeeds "instead of remaining silent or going along with it," as many did in the Majestic case.
"Sometimes police officers are going to cross the line, and we need to hold them accountable, but we don't relish prosecuting law enforcement officers," he said.
Rosenstein said he looks forward to a "culture of integrity" continuing in the department under newly appointed Commissioner Anthony W. Batts, whom Mayor Stephanie Rawlings-Blake selected as Frederick H. Bealefeld III's permanent replacement and who will officially begin Sept. 27 if confirmed by the City Council.


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Sandia National Laboratories on Wednesday shipped its final four truckloads of radioactive legacy waste to the Waste Isolation Pilot Plant near Carlsbad.
Gov. Susana Martinez was on hand for a press conference marking the final shipment of the so-called transuranic waste.
"This is a significant milestone in our efforts to protect New Mexico's environment and keep our state beautiful," Martinez said. "Today's final shipment of TRU waste demonstrates an important partnership with Sandia National Laboratories as we work to preserve our state for future generations."
Following Thursday's press conference, four trucks carrying the final shielded containment vessels of remote handled TRU legacy waste from Sandia National Labs departed from the Eubank Gate heading to WIPP.
The Legacy Waste Clean-up Program is administered by the U.S. Department of Energy and has been set up at several dozen federal lab sites across the country. Both Sandia and Los Alamos National Laboratories in New Mexico participate in the program.
The Legacy Waste program at Sandia began preparation for waste disposal in December 2010. Much of the waste at Sandia was generated as a result of nuclear defense research and weapons manufacturing from programs conducted during the 1970s and 1980s. To date, Sandia has shipped 61 drums of this waste to the Waste Isolation Pilot Plant under the oversight of the New Mexico Environment Department and the Environmental Protection Agency.
"The success of this program is a true testament to the many professionals who have spent years categorizing, separating, and handling the transuranic waste," Martinez said. "I am proud to say that each of these shipments has made it safely to their destination without any associated safety issues.
This achievement is truly reflective of the continued efforts of all those involved in the project."
Martinez also reported today that after the first quarter of this calendar year, Los Alamos National Laboratory is on schedule in their effort to remove above-ground transuranic waste by June of 2014.
This latest effort on the part of LANL is a direct result of the New Mexico Environment Department and Los Alamos National Laboratory entering into a Framework Agreement last fall that calls for all 3,706 cubic meters of above-ground transuranic waste to be removed from the facility by June of 2014.
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Treatment for Migraine Headaches Comes with Positive Side Effect
Neurologists at The Nebraska Medical Center Use Botox to Treat Chronic Migraines
The headaches bothered Tina Bracha for years. The medication helped, but it was not until she started working in the front office of Neurology Consultants of Nebraska that she found out there was another treatment: Botox injections.
"At first I thought, 'there's no way this works,'" Bracha recalled. "But it's been a life-changing thing for me. I don't have headaches every day anymore. I can go outside on these sunny days; I couldn't do that before."
Dr. Santamaria gives Tina Bracha her Quarterly Botox injection Nebraska Medical Center neurologist Pam Santamaria, MD gives Bracha Botox injections once every three months. Each treatment involves 33 shots given in the forehead, temples and back of the neck.
"I've seen it in the clinic and the research bears it out; about 50% of people who don't get significant benefit from the oral medicine will get some benefit from Botox," said Dr. Santamaria. "It either takes the symptoms away completely, or there's some reduction in symptoms."
Botox was approved by the Food and Drug Administration as a migraine treatment in October of 2010. Since then, the number of people seeking the treatment has steadily increased in Dr. Santamaria's clinic.
"Many patients don't realize that Botox is a treatment for migraines," she said. "Most people associate it with cosmetic procedures."
Botox gained popularity as a cosmetic treatment in when it was approved in 2002. That same cosmetic benefit is a side effect of using Botox to treat migraines.
"My kids and husband joke with me," Bracha said. "I have no forehead wrinkles. It's an added benefit, but not having migraines outweighs that. I'd take the wrinkles over the headaches any day."
Because of the Botox treatments, Bracha has neither.
Dr. Santamaria believes migraines are under-treated in the U.S. An estimated 12% of the population deals with migraines and she believes many of those people are simply unaware that treatments exist.
"Migraines are more common than diabetes, high blood pressure, and heart disease," Dr. Santamaria said.
"Migraines are more common in women than men, and occur more in the United States than in other countries."
Not all bad headaches are migraines, Dr. Santamaria said. There is a specific definition that accompanies a migraine diagnosis.
"A migraine is unilateral, meaning it's on one side of the head," she explained. "It's a throbbing, pounding pain that often causes people to have nausea, motion sickness and sensitivity to light."
Those were all symptoms Bracha experienced regularly before her regular Botox treatments.
"When the headaches were really bad I'd miss doing things with my kids," she said. "I had a very hard time just getting out of the house."
Botox works by blocking communication between nerves and muscles, which keeps muscles from contracting. When it is injected in a patient's head, it blocks pain receptors the same way it keeps muscles and nerves from communicating.
Doctors initially discovered the Botox effect on migraines while using it to treat a condition called cervical dystonia. Many who suffer from that condition also struggle with migraines. Patients taking Botox began noticing an improvement of their head ache symptoms. Several years of study followed before the FDA approval came.
"There is really no reason why, if a person is having chronic migraines, not to try Botox as a treatment," said Dr. Santamaria. "It may really help."


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Catalyst Conference
Coding School
Global Classroom
Hacking for Humanity
Get Involved
Mentorship Programs for Women and Girls
The Girls in Tech Mentorship Program focuses on the
, and
of influential women in technology and entrepreneurship. These foci are at the core of our mentorship philosophy, which targets professional women as well as our next generation of women around the world. GIT M takes a three-tiered approach to programming in an effort to create valuable interactions across demographics. Each of these programs is meant to work with each other whenever possible:
GIT W: Professional Women
GIT U: University-aged Women
GIT NX: Girls, our Next Generation
GIT M also highlights the benefits of "reverse mentorship" - learning from those less experienced than yourself - and "sponsorship" - taking a more active approach to mentorship by purposefully creating opportunities and opening doors for mentees. Each program is detailed below.
GIT W: Professional Women
Focused on entrepreneurial/intrapreneurial women who hope to hone their business skills, as well as increase key technical skills needed to advance in the workplace. This program not only includes skill-building elements, but also a developed mentorship network that focuses on leadership, career enhancement and encouragement, while also opening doors and creating advancement opportunities. We have special programs that serve underprivileged populations, providing them access to training and mentorship that increase their opportunities to advance their careers.
GIT U: University-aged Women
GIT U is a program that focuses on attracting and retaining future female talent in the technical fields, providing crucial career advice and networking, and introducing entrepreneurial skills that will benefit students as they enter the professional world. Participating universities develop their own Chapters under the guidance of our Global executive team, and, when possible, alongside existing GIT Chapters. Members can choose to be paired directly with someone in their desired field for one-on-one mentorship opportunities, and can also choose to serve as mentors to our GIT NX constituent.
GIT NX: Girls, our Next Generation
GIT NX focuses on the next generation of girls in technology and STEM fields in an effort to increase knowledge and enthusiasm for the fields, as well as empower young girls who are interested in entering them. Programming includes workshops in various technology and STEM fields – coding, mobile development, engineering, etc – as well as the development of entrepreneurial and leadership skills. Each GIT NX location works closely with existing GIT Chapters for programming, and participants also have the option to be paired with professional women and/or GIT U members for further one-on-one interactions


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A URI is a compact representation of a resource available to your application on the intranet or Internet. The Uri class defines the properties and methods for handling URIs, including parsing, comparing, and combining. The Uri class properties are read-only; to create a modifiable object, use the UriBuilder class.
Relative URIs (for example, "/new/index.htm") must be expanded with respect to a base URI so that they are absolute. The MakeRelative method is provided to convert absolute URIs to relative URIs when necessary.
The Uri constructors do not escape URI strings if the string is a well-formed URI including a scheme identifier.
The Uri properties return a canonical data representation in escaped encoding, with all characters with Unicode values greater than 127 replaced with their hexadecimal equivalents. To put the URI in canonical form, the Uri constructor performs the following steps:
Converts the URI scheme to lowercase.
Converts the host name to lowercase.
If the host name is an IPv6 address, the canonical IPv6 address is used. ScopeId and other optional IPv6 data are removed.
Removes default and empty port numbers.
Canonicalizes the path for hierarchical URIs by compacting sequences such as /./, /../, //, including escaped representations. Note that there are some schemes for which escaped representations are not compacted.
For hierarchical URIs, if the host is not terminated with a forward slash (/), one is added.
By default, any reserved characters in the URI are escaped in accordance with RFC 2396. This behavior changes if International Resource Identifiers or International Domain Name parsing is enabled in which case reserved characters in the URI are escaped in accordance with RFC 3986 and RFC 3987.
As part of canonicalization in the constructor for some schemes, escaped representations are compacted. The schemes for which URI will compact escaped sequences include the following: file, http, https, net.pipe, and net.tcp. For all other schemes, escaped sequences are not compacted. For example: if you percent encode the two dots ".." as "%2E%2E" then the URI constructor will compact this sequence for some schemes. For example, the following code sample shows a URI constructor for the http scheme.
Uri uri = new Uri("http://myUrl/%2E%2E/%2E%2E");
When this code is executed, it returns the following output with the escaped sequence compacted.
The following code example shows a URI constructor for the ftp scheme:
Uri uri = new Uri("ftp://myUrl/%2E%2E/%2E%2E");
When this code is executed, it returns the following output with the escaped sequence not compacted.
You can transform the contents of the Uri class from an escape encoded URI reference to a readable URI reference by using the ToString method. Note that some reserved characters might still be escaped in the output of the ToString method. This is to support unambiguous reconstruction of a URI from the value returned by ToString.
Some URIs include a fragment identifier or a query or both. A fragment identifier is any text that follows a number sign (#), not including the number sign; the fragment text is stored in the Fragment property. Query information is any text that follows a question mark (?) in the URI; the query text is stored in the Query property.
In the .NET Framework version 1.1, if the string specified to a constructor contains an unknown scheme and "c:\", the Uri class inserts "//" after the colon. For example, the URI xyz:c:\abc is converted to xyz://c:/abc. In the .NET Framework version 2.0, this behavior has been removed, and the example string is converted to xyz:c:/abc.
The URI class supports the use of IP addresses in both quad-notation for IPv4 protocol and colon-hexadecimal for IPv6 protocol. Remember to enclose the IPv6 address in square brackets, as in http://[::1].
International Resource Identifier Support
Web addresses are typically expressed using uniform resource identifiers that consist of a very restricted set of characters:
Upper and lower case ASCII letters from the English alphabet.
Digits from 0 to 9.
A small number of other ASCII symbols.
The specifications for URIs are documented in RFC 2396, RFC 2732, RFC 3986, and RFC 3987 published by the Internet Engineering Task Force (IETF).
With the growth of the Internet, there is a growing need to identify resources using languages other than English. Identifiers which facilitate this need and allow non-ASCII characters (characters in the Unicode/ISO 10646 character set) are known as International Resource Identifiers (IRIs). The specifications for IRIs are documented in RFC 3987 published by IETF. Using IRIs allows a URL to contain Unicode characters.
The existing Uri class has been extended in .NET Framework v3.5, 3.0 SP1, and 2.0 SP1 to provide IRI support based on RFC 3987. Current users will not see any change from the .NET Framework 2.0 behavior unless they specifically enable IRI. This ensures application compatibility with prior versions of the .NET Framework.
To enable support for IRI, the following two changes are required:
Add the following line to the machine.config file under the .NET Framework 2.0 directory
Specify whether you want Internationalized Domain Name (IDN) parsing applied to the domain name and whether IRI parsing rules should be applied. This can be done in the machine.config or in the app.config file. For example, add the following:
Enabling IDN will convert all Unicode labels in a domain name to their Punycode equivalents. Punycode names contain only ASCII characters and always start with the xn-- prefix. The reason for this is to support existing DNS servers on the Internet, since most DNS servers only support ASCII characters (see RFC 3940).
Enabling IRI and IDN affects the value of the Uri..::.DnsSafeHost property. Enabling IRI and IDN can also change the behavior of the Equals, OriginalString, GetComponents, and IsWellFormedOriginalString methods.
There are three possible values for IDN depending on the DNS servers that are used:
idn enabled = All
This value will convert any Unicode domain names to their Punycode equivalents (IDN names).
idn enabled = AllExceptIntranet
This value will convert all Unicode domain names not on the local Intranet to use the Punycode equivalents (IDN names). In this case to handle international names on the local Intranet, the DNS servers that are used for the Intranet should support Unicode name resolution.
idn enabled = None
This value will not convert any Unicode domain names to use Punycode. This is the default value which is consistent with the .NET Framework 2.0 behaviour.
Enabling IRI parsing (iriParsing enabled = true) will do normalization and character checking according to the latest IRI rules in RFC 3986 and RFC 3987. The default value is false and will do normalization and character checking according to RFC 2396 and RFC 2732 (for IPv6 literals).
IRI and IDN processing in the Uri class can also be controlled using the System.Configuration..::.IriParsingElement , System.Configuration..::.IdnElement , and System.Configuration..::.UriSection configuration setting classes. The System.Configuration..::.IriParsingElement setting enables or disables IRI processing in the Uri class. The System.Configuration..::.IdnElement setting enables or disables IDN processing in the Uri class. The System.Configuration..::.IriParsingElement setting also indirectly controls IDN. IRI processing must be enabled for IDN processing to be possible. If IRI processing is disabled, then IDN processing will be set to the default setting where the .NET Framework 2.0 behavior is used for compatibility and IDN names are not used.
The configuration setting for the System.Configuration..::.IriParsingElement and System.Configuration..::.IdnElement will be read once when the first System..::.Uri class is constructed. Changes to configuration settings after that time are ignored.
The System..::.GenericUriParser class has also been extended to allow creating a customizable parser that supports IRI and IDN. The behavior of a System..::.GenericUriParser object is specified by passing a bitwise combination of the values available in the System..::.GenericUriParserOptions enumeration to the System..::.GenericUriParser constructor. The GenericUriParserOptions..::.IriParsing type indicates the parser supports the parsing rules specified in RFC 3987 for International Resource Identifiers (IRI). Whether IRI is used is dictated by the configuration values previously discussed.
The GenericUriParserOptions..::.Idn type indicates the parser supports Internationalized Domain Name (IDN) parsing (IDN) of host names. Whether IDN is used is dictated by the configuration values previously discussed.
Performance Considerations
If you use a Web.config file that contains URIs to initialize your application, additional time is required to process the URIs if their scheme identifiers are nonstandard. In such a case, initialize the affected parts of your application when the URIs are needed, not at start time.
Notes to Callers:
Because of security concerns, your application should use caution when accepting Uri instances from untrusted sources and with dontEscape set to true.You can check a URI string for validity by calling the IsWellFormedOriginalString method.
Windows Mobile for Pocket PC, Windows Mobile for Smartphone, Windows CE Platform Note:
The .NET Compact Framework does not differentiate between relative and absolute paths. Also, the .NET Compact Framework processes URI strings prefixed by the file:// scheme differently from the full .NET Framework. A relative file://myfile specification resolves as \\myfile. Using file:///myfile (three slashes) resolves as \myfile in the root directory. To ensure successful operations, specify absolute path information.
The following example creates an instance of the Uri class and uses it to create a WebRequest instance.
Visual Basic
Dim siteUri As New Uri("http://www.contoso.com/")
Dim wr As WebRequest = WebRequest.Create(siteUri)
Uri siteUri = new Uri("http://www.contoso.com/");
WebRequest wr = WebRequest.Create(siteUri);
Visual C++
Uri^ siteUri = gcnew Uri( "http://www.contoso.com/" );
WebRequest^ wr = WebRequest::Create( siteUri );
var siteUri : Uri = new Uri("http://www.contoso.com/");
var wr : WebRequest = WebRequest.Create(siteUri);
uniform resource identifier
Thursday, July 30, 2009
Posted by Usama Wahab Khan at 9:42 PM
Labels: C#, Silverlight, vb.net
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