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Exploiting sulphur-carrier proteins from primary metabolism for 2-thiosugar biosynthesis

Sulphur is an essential element for life and exists ubiquitously in living systems1,2. Yet, how the sulphur atom is incorporated in many sulphur-containing secondary metabolites remains poorly understood. For C-S bond formation in primary metabolites, the major ionic sulphur sources are the protein-persulphide and protein-thiocarboxylate3,4. In each case, the persulphide and thiocarboxylate group on these sulphur-carrier (donor) proteins are post-translationally generated through the action of a specific activating enzyme. In all bacterial cases reported thus far, the genes encoding the enzyme that catalyzes the actual C-S bond formation reaction and its cognate sulphur-carrier protein co-exist in the same gene cluster5. To study 2-thiosugar production in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action appear similar to those of ThiG, the enzyme catalyzing thiazole formation in thiamin biosynthesis6,7. However, no sulphur-carrier protein gene could be located in the BE-7585A cluster. Subsequent genome sequencing revealed the presence of a few sulphur-carrier proteins likely involved in the biosynthesis of primary metabolites, but surprisingly only a single activating enzyme gene in the entire genome of A. orientalis. Further experiments showed that this activating enzyme is capable of adenylating each of these sulphur-carrier proteins, and likely also catalyzing the subsequent thiolation taking advantage of its rhodanese activity. A proper combination of these sulphur delivery systems is effective for BexX-catalyzed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. These studies represent the first complete characterization of a thiosugar formation in nature and also demonstrate the receptor promiscuity of the sulphur-delivery system in A. orientalis. Our results also provide evidence that exploitation of sulphur-delivery machineries of primary metabolism for the biosynthesis of sulphur-containing natural products is likely a general strategy found in nature.

orientalis. Further experiments showed that this activating enzyme is capable of adenylating each of these sulphur-carrier proteins, and likely also catalyzing the subsequent thiolation taking advantage of its rhodanese activity. A proper combination of these sulphur delivery systems is effective for BexX-catalyzed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. These studies represent the first complete characterization of a thiosugar formation in nature and also demonstrate the receptor promiscuity of the sulphur-delivery system in A. orientalis. Our results also provide evidence that exploitation of sulphur-delivery machineries of primary metabolism for the biosynthesis of sulphur-containing natural products is likely a general strategy found in nature.
Unusual sugars found in many secondary metabolites play crucial roles in determining the efficacy and specificity of the biological activities of the parent molecules 8,9 . Despite recent advances made in unusual sugars biosynthesis research, little is known about thiosugar formation due to their rarity in natural products and the limited knowledge of sulphur incorporation in secondary metabolites 1,2,10,11 . In our studies of the biosynthesis of 2thiosugar-containing antibiotic BE-7585A (1, Fig. 1a) in A. orientalis subsp. vinearia BA-07585, we identified a putative 2-thioglucose-6-phosphate synthase, BexX 6,7 , having significant sequence homology to thiazole synthase, ThiG 12 , which is responsible for the construction of thiazole moiety (8) from DXP (6) in thiamin biosynthesis ( Fig. 1b) 13,14 .
Since ThiG catalyzes sulphur insertion into a ThiG-ketosugar adduct (7) 12 , BexX may play a similar role in the conversion of glucose-6-phosphate (G6P, 3) to 2-thioglucose (2) in A. orientalis (Fig. 1a). The proposed function of BexX is supported by the detection of a covalent adduct (4) between BexX and a 2-ketosugar derived from G6P 7 . The crystal structure of BexX-substrate complex has now been determined to 2.3 Å resolution (Extended Data Fig. 1) confirming that the covalent attachment of G6P is at Lys110 of BexX (Fig. 1c). However, the absence of genes encoding potential sulphur transfer enzymes, including common sulphur-carrier proteins 15 , cysteine desulphurases 16 , and rhodanese-like proteins 17 , in and around the BE-7585A biosynthetic gene cluster impeded further functional characterization of BexX.
To search for the sulphur-carrier protein required for BexX reaction, the entire genome of A. orientalis was sequenced. A total of 9,210-coding open reading frames in approximately 9.8 Mb genomic DNA was identified, including genes encoding five cysteine desulphurase homologues, five rhodanese homologues, and four sulphur-carrier protein homologues (AoThiS, AoMoaD, AoCysO and AoMoaD2) (Extended Data Table 1). The thiS, moaD and cysO genes are part of the thiamin, molybdopterin and cysteine biosynthetic gene clusters, respectively 18,19 , whereas moaD2 stands alone with no nearby genes related to any biosynthetic pathway (Extended Data Fig. 2a-d and Supplementary Table 1). While the protein receptor of AoMoaD2 is not immediately apparent, it likely functions as a MoaD homologue due to its high sequence identity to MoaD. In view of the sequence similarity between BexX and ThiG and their mechanistic parallels 7 , we anticipated that AoThiS, being the cognate sulphur-carrier partner of ThiG 13,14 , might be recruited for sulphur delivery to the BexX-G6P complex (4) in A. orientalis.
However, unlike the thiamin biosynthetic gene clusters in Escherichia coli and Bacillus subtilis 13,14 , the one in A. orientalis does not contain thiF, which encodes the ThiS activating enzyme that is essential for converting ThiS to its thiocarboxylate form (10). The corresponding activating enzymes for AoMoaD and AoCysO are also missing from the respective molybdopterin and cysteine biosynthetic gene clusters. To our surprise, only a single putative activating enzyme is found in the entire genome of A. orientalis: a MoeZ homologue consisting of a ThiF/MoeB-like domain at the N-terminal and a rhodanese homology domain at the C-terminal (Extended Data Fig. 2 and Supplementary Table 1). Due to its unique occurrence in the genome and because it is not part of the molybdopterin gene cluster or associated with any other biosynthetic gene cluster, this protein, AoMoeZ, may be the universal activating catalyst for thiocarboxylate protein production in A. orientalis (Fig.  2a).
To test the proposed function of AoMoeZ, the AoThiS and AoMoeZ of A. orientalis were heterologously expressed in E. coli, each with an N-terminal His 6 -tag. When AoThiS was incubated with AoMoeZ and ATP, an ESI-MS signal corresponding to the adenylated AoThiS (9) was detected along with few peaks likely derived from reaction of the labile adenylated AoThiS with buffer components (Extended Data Fig. 3c). Upon the addition of excess bisulphide, complete conversion of 9 to its thiocarboxylate form (10) was observed (Extended Data Fig. 3d). Control experiment using bisulphide in the absence of AoMoeZ showed no change of the original AoThiS signals. These results demonstrated that AoMoeZ is capable of charging AoThiS to its ready-to-use thiocarboxylate form (Fig. 2a). The activated AoThiS-COSwas next incubated with the BexX-G6P complex (4). If sulphur transfer occurs and the resulting 2-thiosugar product (5) is released from the enzyme, a shift of the mass signal corresponding to the BexX-G6P complex to that of the free enzyme is anticipated (Fig. 2b). However, no increase of the free BexX in the presence of AoThiSCOS − was observed (Fig. 2c-e), and hence ruled out AoThiS (and bisulphide) as the sulphur donor for BexX in 2-thiosugar formation.
To assess the competence of AoMoaD, AoCysO and AoMoaD2 as sulphur-carrier proteins in BexX-catalyzed reaction, the N-His 6 -tagged AoMoaD, AoCysO and AoMoaD2 were prepared. Similar to the case of AoThiS, thiocarboxylation of each protein in the presence of AoMoeZ, ATP and NaSH was confirmed by MS analysis (Extended Data Fig. 3f-k). Since the activated AoMoaD was generated in low quantity and purity, only AoCysO and AoMoaD2 were used in the incubation with the BexX-G6P complex. Relative intensities of mass signals corresponding to BexX-G6P (4) and the free enzyme were monitored before and after the addition of the activated sulphur-carrier proteins. To our delight, only signal ascribed to free BexX was discernible after treatment with AoCysO and AoMoaD2 (Fig. 2f, g). To gain further evidence, the thiosugar product (5) was derivatized with monobromobimane (mBBr) prior to HPLC analysis to give 11 to facilitate detection (Fig.  2b). Indeed, when AoCysO or AoMoaD2 was used, a clear appearance of a new peak (product peak) along with the increase in AMP production was observed (Fig. 2h, trace 3, 4). The product peak was isolated and characterized as 2-thioglucose-6-phosphate-bimane (11) by ESI-MS and NMR (Supplementary Information). Each assay sample was also treated with alkaline phosphatase, and the dephosphorylated product matches well with the synthetic standard (Extended Data Fig. 4). As expected, no thiosugar product was detected in the sample of AoThiS. These results firmly established that BexX-catalyzed 2-thiosugar formation could proceed in the presence of either AoCysO-COS − or AoMoaD2-COS − , but not AoThiS-COS − . These two examples clearly reveal the moonlighting capability of some of the sulphur-delivery enzymes in natural product biosynthesis and nicely bridge the biosynthetic pathways of primary and secondary metabolites.
Thiocarboxylated sulphur-carrier proteins recognize their partners through specific protein/ protein interactions. [20][21][22] Because BexX shares 37% sequence identity with ThiG 22 and the two enzymes are structurally homologous, it was surprising to find that AoThiS does not serve as a sulphur-carrier protein for BexX. To understand the sulphur-carrier protein specificity of BexX, the BexX/AoCysO structure was determined to 2.6 Å resolution (Fig.  3a, Extended Data Fig. 1f). We were unable to crystallize BexX/AoMoaD2; however, because of the compact ubiquitin-like fold and similar sizes of AoCysO (90 residues) and AoMoaD2 (96 residues), we were able to construct a reliable homology model for BexX/ AoMoaD2 using the BexX/AoCysO structure as a template. We also constructed a hypothetical model of BexX/AoThiS using ThiS from Thermus thermophilus (PDB code: 2HTM) as a guide. AoCysO and AoMoaD2 superimpose very well with a root mean square deviation (rmsd) of 0.1 Å for 80 Cα carbon atoms. In contrast, AoCysO and AoThiS show significant differences, especially in the loop regions, with an rmsd of 2.7 Å for 43 Cα carbon atoms (Fig. 3b, Extended Data Fig. 1d, e and 5). The most significant difference between either AoCysO or AoMoaD2, and AoThiS (66 residues) is the insertion of two additional α-helices, which are located in the BexX/sulphur-carrier protein interface (Extended Data Fig. 5). As a result the amount of accessible surface area buried upon complex formation is ~1000 Å 2 for BexX/AoCysO and BexX/AoMoaD2 compared to only ~600 Å 2 in BexX/AoThiS (Extended Data Fig. 5, 6a). AoCysO contributes 19 residues and BexX contributes 26 interface residues to the interface, which is similar to 16 AoMoaD2 residues and 23 BexX residues in BexX/AoMoaD2. In contrast, only eight AoThiS residues contribute to the interface in the BexX/AoThiS model. Ten interface residues are conserved between AoCysO and AoMoaD2, but only four of these are conserved in AoThiS (Extended Data Fig. 5a). The hydrogen bonding scheme for BexX/AoCysO is also conserved in BexX/ AoMoaD2 (Extended Data Fig. 6b-d). A comparison of the BexX/AoCysO complex and the Bacillus subtilis ThiG/ThiS (PDB ID: 1TYG) 22 complex provides further insight. Superposition of BexX/AoCysO and ThiG/ThiS results in an rmsd of 1.7 Å for the BexX-ThiG core (Fig. 3c); however, AoCysO and ThiS do not overlay well (rmsd more than 40 Å). Thus, even though the overall sulphur-carrier protein folds are similar, and each sulphurcarrier protein is positioned to insert its C-terminal tail into the active site of its partner (Extended Data Fig. 6e, f), the selection of AoCysO or AoMoaD2 by BexX is clearly determined by the interface interactions.
Finally we also examined whether the C-terminal rhodanese domain of AoMoeZ plays a role in sulphur transfer. In a typical rhodanese reaction, the conserved cysteine residue in rhodanese (e.g., C360 in AoMoeZ) is converted to a persulphide group in the presence of thiosulphate or through the action of a cysteine desulphurase using L-cysteine as the sulphur source 17 . Since the resulting persulphide is a known sulphur donor 3 , it can be used to charge the adenylated sulphur-carrier proteins to the thiocarboxylate forms (Fig. 4). To test the potential second role of AoMoeZ as a sulphur donor, incubation of AoMoeZ and AoCysO or AoMoaD2 was first carried out in the presence of ATP and thiosulphate (with no addition of reducing agent to prevent bisulphide formation). In both cases, thiolation of AoCysO and AoMoaD2 catalyzed by AoMoeZ was observed but not with the AoMoeZ(C360A) mutant (Extended Data Fig. 7) which retained a similar level of adenylation activity (Extended Data Fig. 8). These observations are consistent with the involvement of the C-terminal rhodanese domain of AoMoeZ in sulphur transfer. Next, BexX-catalyzed 2-thiosugar formation was performed with AoMoeZ and AoMoaD2 in the presence of ATP using either thiosulphate ( Fig. 4a) or L-cysteine and a cysteine desulphurase (CD4, Supplementary Table S2) from A. orientalis (Fig. 4b) as the primary sulphur sources. As expected, the 2-thioglucose product was detected in both cases in the absence of reducing agents (Extended Data Fig. 9). Taken together, these results support the likely dual role of AoMoeZ in catalyzing both adenylation and thiolation of the sulphur-carrier proteins.
In summary, we have performed whole genome sequencing of A. orientalis and demonstrated that sulphur delivery for 2-thiosugar production in the biosynthesis of BE-7585A is achieved by hijacking sulphur transfer systems from primary metabolism. Although the overall reaction mechanism of 2-thiosugar formation resembles that of thiamin biosynthesis, BexX cannot utilize the corresponding sulphur-carrier protein, AoThiS, from the thiamin pathway. Instead, sulphur-carrier proteins likely involved in cysteine (AoCysO) and molybdopterin (AoMoaD2) biosynthesis are recruited to transfer their C-terminal thiocarboxylate sulphur to the BexX-G6P complex (4). Two structural snap shots, which represent the BexX-G6P ketone intermediate (4) and the BexX/AoCysO heterotetramer, provide significant insights into the proposed sulphur incorporation mechanism as well as the structural basis by which sulphur-carrier proteins are selected. These results indicate that a functional alliance between a sulphur-carrier protein and its acceptor protein is not specific, but is not entirely random. The assembly of operational sulphur transfer machinery by using components from sulphur-carrier systems of primary metabolism to deliver sulphur atom to make 2-thiosugar represents an efficient strategy for the biosynthesis of a relatively rare metabolite. Such an ad hoc approach of sulphur transfer may represent a paradigm for as yet undiscovered pathways of sulphur-containing natural product biosynthesis. The revelation that AoMoeZ is the universal activating enzyme for all known sulphur-carrier proteins in A. orientalis is another significant finding. The unique presence of only a single ThiF-type enzyme in the entire genome has also been noted in several other microorganisms (Extended Data Table 2). It appears that the charging of multiple sulphur-carrier proteins in different biosynthetic pathways by one activating-enzyme may be a common phenomenon in nature (at least in Actinomycetales) 5,23,24 . In addition, the fact that functional pairs of sulphur-carrier proteins and their acceptor proteins are not necessarily located in the same gene cluster raises the possibility that some cryptic gene clusters in various genomes may actually encode pathways for the biosynthesis of sulphur-containing natural products. Such a possibility has generally been overlooked in current efforts to deconvolute genomic information.

Whole-genome sequencing and analysis
Spores of A. orientalis were inoculated into 10 mL of soybean-casein digest (TSB) medium and grown in a rotary incubator at 30 °C and 250 rpm for 2 days. The resultant seed culture (4 mL) was transferred to 100 mL of TSB medium and grown under the same conditions for 2 days. The growth culture (25 mL) was centrifuged at 5,000 × g for 20 min at 4 °C, and the cells were washed with 25 mL of 10 mM EDTA. After another centrifugation, the cells were stored at −80 °C until use. The cells were resuspended in 5 mL of 1 mM EDTA, and the suspension was divided into 1.5 mL-tubes (0.4 mL each). The genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (Invitrogen) according to the manufacturer's instructions. The resultant DNA solution (0.5 μg/μL, 50 μL from each tube) was subjected to massive parallel sequencing using Roche 454 GS FLX Titanium and Illumina Genome Analyzer IIx at the High Throughput Sequencing Core Facility of the Academia Sinica in Taiwan. Primary assembly was carried out using Roche 454 Newbler software. Contig extension and closing of short gaps was achieved by scripts built in-house at the core. Genome annotation was performed using Glimmer (Gene Locator and Interpolated Markov ModelER) version 3.0 25 , tRNAscan-SE 26 , and RNAmmer 27 . Homologous protein sequences were identified in the NCBI database using the basic local alignment search tool (BLAST).

Preparation of proteins
C-His 6 -BexX was prepared as described before. 7 The thiS (orf13974), moaD (orf13839), cysO (orf06461), moaD2 (orf10102), moeZ (orf02110), and cd4 (orf04763) genes were PCR-amplified from A. oreintalis genomic DNA using primers with engineered NdeI and HindIII restriction sites. The sequences of the primers are described in the Supplementary information. The PCR-amplified gene fragments were purified, digested with NdeI and HindIII and ligated into pET28b(+) vector (Novagen) that was also digested with the same enzymes. For crystallization studies, the bexX gene was also subcloned into pET28b(+) and produced as an N-terminal His 6 -tagged protein. In addition, the cysO gene was subcloned into IMPACT™pTYB1 vector (New England Biolab) digested with NdeI and SapI for production of AoCysO thiocarboxylate. 28 The resultant plasmids were used to transform E. coli BL21 star (DE3) strain (Invitrogen) for protein overexpression. An overnight culture of E. coli transformants grown in the LB medium (10 mL) containing 50 μg/mL of kanamycin at 37 °C were used to inoculate 1 L of the same growth medium. The culture was incubated at 37 °C with shaking (230 rpm) until the OD 600 reached ~0.5. Protein expression was then induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and the cells were allowed to grow at 18 °C and 125 rpm for an additional 24 h. The cells were harvested by centrifugation at 4,500 × g for 15 min and stored at -80 °C until lysis. All purification steps were carried out at 4 °C using Ni-NTA resin according to the manufacturer's protocol. The proteins were eluted using 250 mM imidazole buffer containing 10% glycerol except those for crystallization studies. The collected protein solution was dialyzed against 3 × 1-L of 50 mM Tris·HCl buffer (pH 8) containing 300 mM NaCl and 15% glycerol. The protein solution was then flash-frozen in liquid nitrogen and stored at −80 °C until use. For crystallization studies, N-His 6 -BexX eluted with 250 mM imidazole buffer was incubated with 2 mM glucose 6-phophate (G6P) and 2 mM dithiothreitol for 1 h at 4 °C and then further purified in 10 mM Tris·HCl buffer (pH 8.0) containing 50 mM NaCl by using a Superdex G200 column (GE Healthcare). In case of cysO/pTYB1, the cell lysate were loaded onto a column of chitin beads (New England Biolabs, 10 mL) at a flow rate of 0.8 mL/min. The column was then washed with 15 column volumes of column buffer at a flow rate of 2 mL/min. Intein mediated cleavage of the protein was carried out at 18 °C for 12 h with 30 mL of 50 mM dithiothreitol to yield AoCysO or with Na 2 S to yield AoCysO-thiocarboxylate. 28 AoCysO was further purified in 50 mM NaCl, 10 mM Tris·HCl buffer (pH 8.0) containing 50 mM NaCl by using a Superdex G75 column (GE Healthcare). Protein concentration was determined by the Bradford assay using bovine serum albumin as the standard. 29 The molecular mass and purity (> 90% except N-His 6-AoMoaD) of the proteins were estimated by SDS-PAGE analyses (Extended Data Fig. 3l).

2-Thiosugar Formation and its Detection
The typical BexX reaction mixture (50 μL) contained C-His 6 -BexX (100 μM), sulphur carrier proteins (N-His 6 -AoCysO (30 μM), NHis 6 -AoMoaD2 (45 μM), or N-His 6 -AoThiS (45 μM)), N-His 6 -AoMoeZ (15 μM), ATP (2 mM), glucose-6-phosphate (G6P, 2 mM), NaSH (5 mM) in 50 mM NH 4 ·HCO 3 buffer (pH 8.0) containing MgCl 2 (5 mM). The resulting reaction mixture was incubated at room temperature for 8 h and stored at −20 °C until analysis. To the reaction mixture (10 μL) prepared above was then added 10 μL of 10 mM monobromobimane (mBBr) methanol solution (final concentration of mBBr was 5 mM) and incubated at room temperature for 5 min. The mixture was centrifuged at 16,000 × g for 5 min to remove the precipitant, and 10 μL of the supernatant was transferred to a new tube. The solution was evaporated in vacuo using a Speedvac SC100 (Savant). The resulting residue was redissolved in 100 μL of 50 mM NH 4 ·HCO 3 buffer (pH 8.0) and subjected to HPLC analysis using a Dionex CarboPac PA1 analytical column (4 × 250 mm). The sample was eluted with a gradient of water (solvent A) and 1 M NH 4 OAc (solvent B). The gradient was run from 5 to 15% B over 5 min, 15−30% B over 15 min, 30−100% B over 7 min with a 5 min wash at 100% B, and 100−5% B over 3 min, followed by re-equilibration at 5% B for 5 min. The flow rate was 1 mL/min, and the detector was set at 260 nm (Fig. 2h). The peak corresponding to the enzymatic reaction product was isolated and subjected to ESI-MS and NMR analyses (see Supplementary information for the structural characterization). Alternatively, the reaction mixture stored at −20 °C was thawed and treated with calf intestinal alkaline phosphatase (CIP) (0.2 μL, 2 units) and incubated at 37 °C for 1 h. The precipitant that appeared during the incubation was removed by centrifugation at 16,000 × g for 2 min, and 2 μL of a 100 mM monobromobimane (mBBr) methanol solution was added to the reaction solution (final concentration of mBBr was 5 mM). The resulting mixture was incubated at room temperature for 5 min, and the supernatant (5 μL) was diluted with deionized water (95 μL) prior to HPLC analysis using an analytical C 18 column (4 × 250 mm). The sample (20 μL) was eluted with a gradient of water (solvent A) and 80% acetonitrile (solvent B). The gradient was run from 5 to 30% B over 15 min, 30−80% B over 5 min, 80−5% B over 5 min, followed by reequilibration at 5% B for 10 min. The flow rate was 1 mL/min, and the detector was set at 260 nm. The 2-thio-D-glucose-bimane standard (0.1 mM) was prepared from the chemically synthesized 2-thio-D-glucose 6 incubated with mBBr at room temperature for 5 min. The peak corresponding to the enzymatic reaction product was also isolated and subjected to ESI-MS analysis (Extended Data Fig. 4).

Determination of Rhodanese Activity of AoMoeZ
The site-specific C360A mutant of AoMoeZ were constructed according to the mutagenesis protocol using moeZ/pET28b(+) as DNA template. The forward primer 5'-GATCGTCCTGCACGCCAAGTCGGGC-3' and the reverse primer 5'-GCGGGCGCCCGACTTGGCGTGCAGG-3' were used in the PCR amplification. The resulting plasmids moeZ(C360A)/pET28b(+) were used to transform the E. coli BL21 star (DE3) strain for protein overexpression. The rhodanese activity of AoMoeZ was determined using the assay developed by Sörbo 30 . A typical assay mixture contained 50 mM Tris·HCl (pH 8.0), 50 mM KCN, approximately 2 μM of AoMoeZ or AoMoeZ(C360A), and a variable amount of sodium thiosulfate (from 0 to 35 mM) in a volume of 100 μL. The reaction was initiated by the addition of AoMoeZ and was quenched after 10-s incubation at 25 °C by the addition of 50 μL of reagent A (15% formaldehyde). Then 150 μL of reagent B (1 g Fe(NO 3 ) 3 ·9H 2 O and 2 mL 65% HNO 3 in 13 mL H 2 O) was added for color development. Formation of SCN − in the reaction was quantified using the extinction coefficient for Fe(SCN) 3 (4200 M −1 cm −1 at 460 nm). The steady state kinetic parameters were determined in triplicates by fitting the experimental data using Michaelis-Menten equation (Extended Data Fig. 7e). Assay for protein thiocarboxylate formation was performed in the anaerobic chamber to minimize the oxidation of AoMoeZ or AoMoeZ(C360A). A typical reaction mixture contained 80 μM AoMoeZ or AoMoeZ(C360A), 4 mM ATP, 5 mM MgCl 2 , 5 mM Na 2 S 2 O 3 in 50 mM HEPES (pH 8.0) with 500 mM glycerol (from enzyme stock solution), and 100 μM of one of the sulphur carrier proteins, N-His 6 -AoMoaD2 or N-His 6 -AoCysO. The reaction was incubated in the glove-box at ~30 °C for 40 min and then quenched by flash freezing in liquid nitrogen. Samples were then submitted to ESI-MS for analysis (Extended Data Fig. 7a-d).

Spectrophotometric analysis of the adenylation reaction catalyzed by AoMoeZ and its C360A mutant
The adenylation of sulphur-carrier proteins catalyzed by MoeZ and its C360A mutant was monitored using a coupled enzyme assay in the presence of an excess of NaSH (Extended Data Fig. 8a). The coupled enzyme reaction was monitored by detecting the consumption of NADH (ε 340 = 6220 M −1 cm −1 ) at 340 nm. A typical reaction mixture (120 μL) contained AoMoeZ or its C360A mutant (3 μM), MoaD2 (10 μM), ATP (80 μM), NaSH (3 mM), phosphoenolpyruvate (PEP, 2 mM), NADH (0.16 mM), and ~1.5 unit each of adenylate kinase (AK), pyruvate kinase (PK), and lactate dehydratase (LDH) in 50 mM NH 4 ·HCO 3 buffer (pH 8.0) containing MgCl 2 (2.5 mM). The reaction was initiated upon addition of ATP at time 0, and absorbance at 340 nm was monitored for 30 sec (Extended Data Fig. 8b). . After incubation at room temperature for 30 min, the reaction mixture was dried by speed vacuum. The residue was re-dissolved in 50 mM NH 4 ·HCO 3 (pH 8.0) and treated with 3 unit of CIP at 37 °C for 1.5 h. The CIP enzyme was removed by centrifugation after precipitation with acetonitrile (50% v/v in final concentration), and the collected supernatant was dried by speed vacuum. The residue was then dissolved in 40 µL of deionized water prior to HPLC analysis with a C 18 column (4 × 250 mm). Each sample (20 μL) was eluted with a gradient of water (solvent A) and acetonitrile (solvent B). The gradient was run from 4 to 24% B over 15 min, 24−64% B over 5 min, 64−4% B over 5 min, followed by re-equilibration at 5% B for 8 min. The flow rate and the detector setting were the same as described above (1 mL/min, 260 nm). The 2-thio-D-glucose-bimane standard (10, 25, 50, 77, 100, 200 μM) described above was also injected into HPLC for calibration of the peak area (Extended Data Fig. 9).

Crystallization of BexX-G6P
Crystals of BexX-G6P were grown using the vapor diffusion hanging drop method. A solution containing 10 mg/mL of BexX in 10 mM Tris, 50 mM NaCl, pH 8.0 was preincubated on ice with G6P at final concentration of 2 mM for about 1 h. Hanging drops were formed by mixing 1.5 μL of protein solution and 1.5 μL of well solution containing 40% (v/v) PEG300, 0.1 M sodium cacodylate/HCl, pH 6.5 and 0.2 M calcium acetate. Rod shape crystals grew in about 6 days to their maximum size of 0.2-0.3 mm × 0.1-0.2 mm. Preliminary X-ray analysis showed that the crystals belonged to the space group P4 1 2 1 2 or P4 3 2 1 2 with unit cell dimensions of a = 168.9 Å and c = 42.4 Å. The Matthew's coefficient assuming two monomers of BexX per asymmetric is 2.8 Å 3 /Da and corresponds to a solvent content of 56.1%.

Crystallization of BexX/AoCysO Complex
Crystals of BexX/AoCysO were grown using the vapor diffusion hanging drop method. Both AoCysO and AoCysO thiocarboxylate were used for crystallization trials; however, AoCysO consistently gave better crystals and was used for the structures reported here. The BexX/AoCysO complex was formed by preincubating 0.55 mL of BexX at 15 mg/mL and 1.0 mL of AoCysO at 10 mg/mL in 10 mM Tris, 50 mM NaCl, pH 8.0 for 1 h. Hanging drops were formed by mixing 1.5 μL of protein solution and 1.5 μL of well solution containing 28% PEG4000, 0.1 M Tris, pH 8.0 and 0.2 M LiSO 4 . Plate shape crystals appeared within 5 days and grew to their maximum size of 0.5 mm × 0.4 mm × 0.02 mm in about two weeks. The crystals belonged to space group I422 with unit cell dimensions of a = 106.4 Å and c = 181.7 Å. The Mathew's coefficient is 3.5 Å 3 /Da assuming one monomer of BexX and one monomer of AoCysO per asymmetric unit and corresponds to a solvent content of 64.5%.

X-ray Data Collection and Processing
X-ray diffraction data for BexX-G6P were collected at beamline A1 at the Cornell High Energy Synchrotron Source (CHESS) using an Area Detector Systems Corporation (ADSC) Quantum 210 CCD detector with a crystal to detector distance of 200 mm and a wavelength of 0.9767 Å. The data collection temperature was 100 K. A total of 180° of data was collected with an oscillation range of 0.5° per frame and an exposure time of 3 s per frame. Data for BexX-G6P/AoCysO were collected at NE-CAT beamline 24-ID-C at the Advanced Photon Source (APS) using an ADSC Q315 CCD detector. The wavelength was 0.9791 Å, the data collection temperature was 100 K, and the detector distance was 400 mm. Individual frames were collected over a range of 180° using 1 s for each 1.0°. X-ray diffraction data were indexed, integrated, scaled, and merged using the program HKL2000 31 .

Structure Determination and Refinement
The structure of BexX was determined by molecular replacement using Phaser 32 as implemented in the PHENIX 33 program package. A monomer of thiamin thiazole synthase (ThiG) from Bacillus subtilis (PDB ID: 1TYG) 22 , which shares 37% sequence identity with BexX, was modified using Chainsaw in CCP4 34 to generate a search model. The initial molecular replacement solution was refined to an R-factor of 40.0% and R-free of 44.0%. All side chains were added and the model was manually adjusted using COOT 35 . After several cycles of refinement using PHENIX 33 and Refmac5 36 , G6P and water molecules were added. The final model was refined to an R-factor of 19.1% and R-free of 22.2%. The Ramachandran plot shows 94.7% of residues in the most favorable regions and 5.3% in the allowed regions. No residues were in generously allowed regions or disallowed regions. The structure of BexX/AoCysO was determined by molecular replacement using a monomer of BexX from the BexX-G6P complex and a monomer of CysO from Mycobacterium tuberculosis (PDB ID: 3DWM) 20 as the search models. An initial model, corresponding to one monomer of BexX and one monomer of AoCysO, was generated by Phaser 32 as implemented in PHENIX 33 . Packing analysis showed that a BexX/AoCysO dimer is formed by crystallographic two-fold symmetry. The initial refinement resulted in an R-factor of 33.8% and R-free of 39.5%. Subsequent cycles of model building in COOT 35 and refinement in PHENIX 33 and Refmac5 36 resulted in a final R-factor of 19.6% and R-free of 24.0%. The Ramachandran plot shows that 90.0% of residues are located in the most favorable regions, 9.7% in the allowed regions, and 0.3% in the generously allowed regions. No residues were in the disallowed regions.    (or AoMoaD2) and AoThiS result from an insertion of ten residues between β1 and β2 of AoCysO (or AoMoaD2) and an insertion of 14 (or 15) residues between α1 and β3 of AoCysO (or AoMoaD2). The first insertion includes the short helix 3 10 1 and the second includes helix α2. Both of these insertions are involved in the BexX-AoCysO (or BexX-AoMoaD2) interface. Ten interface residues (marked with red star and red triangle) are conserved between AoCysO and AoMoaD2, however, only four residues are conserved in AoThiS (marked with red triangle). Two differences between AoCysO and AoMoaD2 represent conservative substitutions; while Thr9 and Ala86 in AoCysO is replaced by Gly11 and Ser92 in AoMoaD2, the interface interaction is contributed by hydrogen bonds that formed by the backbone atom. b-d, Hydrophobic interactions of (b) BexX/AoCysO, (c) BexX/ AoMoaD2, and (d) BexX/AoThiS. BexX monomers are shown as gray ribbon diagrams with hydrophobic interaction regions colored in cyan. AoCysO, AoMoaD2 and AoThiS are shown as cartoon and colored in green, skyblue and yellow, respectively. Hydrophobic interaction regions in sulphur carrier protein are colored in red. The α-helices and β-strands in BexX and sulphur carrier proteins are labeled in black and red, respectively. Fig. 6. The BexX/AoCysO interface, predicted hydrogen bonds in BexX with sulphur-carrier proteins, and comparison of the BexX/AoCysO interface to the Bacillus subtilis ThiG/ThiS interface a, Interacting surfaces of BexX (left) and AoCysO (right). The surface is color coded by atom type (oxygen, red; nitrogen, blue; carbon, green). Non-interacting surfaces are color coded gray. b, Hydrogen bonds on the surface of BexX with AoCysO are shown as black dashes. c, Hydrogen bonds formed by the C-terminal tail of AoCysO and the surrounding residues from BexX are shown as black dashes. The Fo-Fc simulated annealing (SA)-omit map of the C-terminal residues (AVAGG) is rendered in gray and contoured at 3.0 sigma. Fig. 8. Relative adenylation activitiy of AoMoeZ and AoMoeZ(C360A) mutant a, Reaction scheme of the AoMoeZ-catalyzed adenylation activity assay. The adenylation activities of AoMoeZ and its C360A mutant were inferred using a colorimetric assay to monitor the production of AMP (indicated by the decrease of NADH at 340 nm) when AoMoeZ or its C360A mutant was co-incubated with a sulphur carrier protein (AoMoaD2) in the presence of ATP, sodium hydrosulphide, adenylate kinase (AK), pyruvate kinase (PK), and lactate dehydratase (LDH). b, Relative adenylation activity of AoMoeZ (open circle) and its C360A mutant (closed circle) as well as no AoMoeZ/AoMoeZ(C360A) control (open square) were measured by the coupled enzyme assay as described above. Little difference of the decrease of absorpotion at 340 nm was observed for AoMoeZ and its C360A mutant (compare to the control with no AoMoeZ), suggesting that mutation at Cys360 had little effect on the adenylation activity of AoMoeZ. Putative cysteine desulphurases, rhodaneses, and sulphur-carrier proteins found in A. orientalis genome.