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Expression, purification and crystallization of a novel metagenome-derived salicylaldehyde dehydrogenase from Alpine soil

Gene-targeted assembly was used to mine a novel salicylaldehyde dehydrogenase from an Alpine soil metagenome. The enzyme was cloned, expressed, purified and crystallized: it is the first metagenome-derived aldehyde dehydrogenase to be crystallized. Analysis of the crystal structure shows that it adopts the standard conformation of the aldehyde dehydrogenase superfamily and a carboxylic acid was found to be a putative ligand of this enzyme.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are aromatic pollutants that are recalcitrant to degradation and therefore tend to accumulate in the ecosystem. These aromatic compounds consist of multiple fused rings, with the most common ones being five-or six-membered rings, which include anthracene, benzo[a]pyrene, naphthalene, phenanthrene and pyrene (Wang et al., 2017). These hydrophobic compounds are ubiquitous in the environment and pose serious health hazards since they are toxic, teratogenic, carcinogenic and mutagenic (Lee et al., 2018;Dastgheib et al., 2012).
During the degradation of various aromatic hydrocarbons several metabolic intermediates are found, which include some aromatic aldehydes and their derivatives. Noteworthy is salicylaldehyde, which is a key intermediate in naphthalene, phenanthrene, acenaphthene and carbaryl degradation pathways (Ghosal et al., 2016;Mallick et al., 2011). Aldehydes are also vital intermediates in the metabolism of macromolecules and xenobiotics. Although aromatic and aliphatic aldehydes are extensively used in industry, these compounds have been found to be toxic to life (Caboni et al., 2013;Roy & Das, 2010).
As a key intermediate in the breakdown of some aromatic PAHs, salicylaldehyde can be oxidized to salicylate by the activity of salicylaldehyde dehydrogenase (SALD; EC 1.2.1.65), which is an NAD(P) + -dependent enzyme. In naphthalene degradation, this enzyme catalyses the last reaction of the upper pathway (Seo et al., 2009;Eaton & Chapman, 1992). SALD belongs to the superfamily of NAD(P) + -dependent aldehyde dehydrogenases (ALDHs). Generally, the enzymes of this superfamily catalyse the oxidation of a broad range of aldehydes to their corresponding carboxylic acids, playing a major role in detoxification. Structurally, the scaffold of ALDHs is comparable, in which they possess three domains: an NAD(P) + cofactor-binding domain, a catalytic domain and a bridging domain (Marchler-Bauer et al., 2013;Perozich et al., 1999).
Several studies have reported the in vivo activity of SALDs from a range of aromatic hydrocarbon-degrading microorganisms (Rosselló -Mora et al., 1994;Grund et al., 1992;Schell, 1983), and a few studies have described the purification and characterization of the enzyme (Coitinho et al., 2016;Singh et al., 2014). Only Coitinho et al. (2016) have reported a crystal structure of this enzyme, which they isolated from Pseudomonas putida G7.
In our laboratory, we have recently been engaged in the discovery and characterization of novel enzymes from Alpine metagenomes (Dandare et al., 2019). Here, we report the exploitation of molecular-biology strategies (cloning, heterologous overexpression and protein purification) to obtain the novel Alpine metagenome-derived SALD AP . We further crystallized the enzyme, collected diffraction data and solved its structure. To the best of our knowledge, this is the first report of the crystallization and structure of a metagenomederived ALDH.

Materials and methods
2.1. Macromolecule production 2.1.1. Molecular cloning. Following the discovery of SALD AP by gene-targeted assembly, DNA was isolated from Alpine soil samples  and used as the template for polymerase chain reaction (PCR) to amplify the SALD AP gene. The primers, expression vector and host are given in Table 1.
The DNA fragments obtained on a 1% agarose gel after PCR were excised, purified and inserted into a pLATE51 vector (Thermo Fisher Scientific). The resulting recombinant p51-SALD AP plasmid was then transformed into Escherichia coli BL21 (DE3) chemically competent cells and positive transformants were confirmed by colony PCR and sequencing using the pLATE vector primers.

Protein expression.
A confirmed positive clone was inoculated in 10 ml LB broth supplemented with 100 mg ml À1 ampicillin and grown overnight. A 2 l flask containing 800 ml LB broth supplemented with 100 mg ml À1 ampicillin was then inoculated with 5 ml of the overnight culture of the recombinant p51-SALD AP cells. The large-scale culture was incubated at 30 C with shaking (200 rev min À1 ) until the midexponential phase of growth (OD 600 ' 0.6); it was then induced for protein expression with a final concentration of 1 mM isopropyl -d-1-thiogalactopyranoside and allowed to grow under the same conditions for 6 h.
The bacterial cells were harvested by centrifugation (4 C, 7000g, 30 min) and resuspended in 20 ml lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 5 mM imidazole pH 8.0, 0.2 mg ml À1 lysozyme, 0.5 mM phenylmethylsulfonyl fluoride). Cell lysis was achieved by mechanical disruption using a Soniprep 150 with three successive cycles at an amplitude of 16 mm. Each sonication cycle included 30 s on per pulse on an ice bath to minimize heat accumulation, which could consequently lead to protein degradation. Subsequently, the supernatant containing the soluble protein was separated from the cell debris by centrifugation at 4 C for 30 min at 15 000g.
2.1.3. Purification. The expressed protein possessed an N-terminal 6ÂHis tag; therefore, it was purified by metalaffinity chromatography using HIS-Select cobalt (Co 2+ ) affinity resin. An Econo column was packed with 1 ml of the resuspended resin and equilibrated with four column volumes of equilibration buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole pH 8.0). The supernatant containing the recombinant protein was poured into the column and the  Table 1 Macromolecule-production information.
In the primers, the underlined sequences are the specific flanking sequences required to generate the overhangs necessary for ligation-independent cloning (LIC) of the gene into pLATE51 (p51) vector, which adds an N-terminal 6ÂHis tag to the target protein. The non-underlined sequences represent the SALD AP gene-specific sequences. flowthrough was collected. The column was washed twice in each cycle with four column volumes of equilibration buffer. Finally, 4 ml of elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole pH 8.0) containing a high concentration of imidazole was used to elute the retained His-tagged protein from the affinity resin. Subsequently, the eluted recombinant 6ÂHis SALD AP protein was extensively dialysed against dialysis buffer (20 mM sodium phosphate buffer pH 7.5, 20 mM NaCl). The dialysed protein was further purified using gel filtration on a K 9/30 chromatography column prepacked with Sephacryl S-300 (Pharmacia) with a bed volume (V t ) of 48 ml. Prior to sample loading, the column was equilibrated with gel-filtration buffer (50 mM Tris-HCl, 17 mM Tris base, 150 mM NaCl pH 7.4) at a flow rate of 1 ml min À1 . The void volume (V 0 ) was determined using blue dextran, and the column was then calibrated with standard proteins (-amylase, 200 kDa; bovine serum albumin, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12.4 kDa), which were used to plot a standard curve in order to assess the oligomerization state of SALD AP . Approximately 300 ml of protein sample was loaded onto the column and 1 ml fractions were collected. The presence of protein in each fraction was determined by measuring the absorbance at 280 nm using a 6705 UV-visible spectrophotometer. The fractions with the highest absorbance were pooled and concentrated using an Amicon Ultra-30k centrifugal filter (Millipore) until a final protein concentration of 12 mg ml À1 was obtained, which was used in crystallization trials.

Crystallization
SALD AP at a concentration of 12 mg ml À1 in 20 mM sodium phosphate buffer, 20 mM NaCl pH 7.4 was used in crystallization experiments with 2 mM tris(2-carboxyethyl)phosphine (TCEP) added to keep the protein reduced. Before crystallization experiments, the protein solution was centrifuged at 10 000g at 4 C for 10 min. The crystal was grown from the JCSG+ screen (Molecular Dimensions) at 20 C in a 100 + 100 nl drop set up over 40 ml reservoir consisting of 0.1 M sodium acetate pH 4.6, 8%(w/v) PEG 8000. Table 2 shows a summary of the experimental crystallization setup.
Before flash-cooling in liquid nitrogen, the crystal was dipped into cryosolution consisting of 0.1 M sodium acetate pH 4.6, 10%(w/v) PEG 8000, 25%(v/v) glycerol, 2 mM TCEP. Data were collected from a fragment of a crystal with an original size of about 60 Â 40 Â 10 mm, as seen in Fig. 1.

Data collection and processing
Data were collected to 1.9 Å resolution at 100 K on beamline I03 ( = 1.03865 Å ) at Diamond Light Source using a PILATUS3 6M detector (Dectris). Data were processed using XDS (Kabsch, 2010) and AIMLESS (Evans & Murshudov, 2013). For R free calculations, 5% of the reflections were flagged and were not used for structure refinement.

Structure solution and refinement
The structure was determined with Phaser (McCoy et al., 2007) using a modified model of PDB entry 4jz6 (salicylaldehyde dehydrogenase from P. putida G7 complexed with salicylaldehyde; Coitinho et al., 2016) as a starting model (48% identity to the SALD AP amino-acid sequence). Thereafter, the model was rebuilt using the molecular-graphics software Coot (Emsley et al., 2010) and refined using the reciprocal-space refinement program REFMAC5 (Murshudov et al., 2011) with riding hydrogen atoms, noncrystallographic symmetry (NCS) restraints between the four independent molecules and TLS parametrization (Winn et al., 2001). In the final stages of refinement, BUSTER (Bricogne et al., 2016) was used for refinement. Several ligands were tested and the ligand that best fitted the electron density was used in the final stages of refinement.

Homologous structural comparison
In order to identify proteins that are structurally similar to SALD AP , a protein structure-comparison server (the DALI server; http://ekhidna2.biocenter.helsinki.fi/dali/) was used to perform a three-dimensional search (Holm & Rosenströ m, 2010). Further structural comparisons and analysis of SALD AP and the best structural homologue were carried out by superimposition of the crystal structures using PyMOL version 1.7.4.5.

Differential scanning fluorimetry (DSF)
The thermal stability of the enzyme with and without its ligand(s) was determined using DSF. Initially, the optimum enzyme concentration that gave the best fluorescence signal

Figure 1
The crystal mounted in a nylon loop in the X-ray beam. The red cross indicates the position of the X-ray beam. The red bars indicate the scale and correspond to 100 Â 100 mm.
was determined by enzyme titration (5-7 mM). Also, the optimum concentration of ligand that gave the best signal was determined by measuring different concentrations (0.5-2.0 mM). The assay mixture was made up to a final volume of 20 ml consisting of the enzyme aliquot diluted to the appropriate concentration in 50 mM HEPES pH 7.4. Ligands and cofactor (NAD + ) were added where required. SYPRO Orange (1Â working concentration) was always the last component to be added to the reaction mixture prior to running in the thermocycler. All reactions were prepared on ice to minimize protein denaturation. The reactions were prepared in 0.2 ml PCR tubes in triplicate and were run in a Rotor-Gene Q cycler (Qiagen). A highresolution melt experiment with the following protocol was set up: a temperature rise from 25 to 95 C with a 1 C increase every 5 s without gain optimization. The fluorescence of the protein due to the binding of SYPRO (dye) to its exposed hydrophobic regions as it denatures with increasing temperature was exploited by exciting the enzyme at 460 nm and measuring the emission at 510 nm. This assay was used as a measure of the thermal stability of the enzyme in the presence and absence of ligands.
First-derivative (ÁF/ÁT) plots of the melting curves of the enzyme were used to determine the melting temperature (T m ) of the enzyme. T m is the temperature at which the ÁF/ÁT peak appears. The Rotor-Gene inbuilt analysis software was used to calculate the derivative of fluorescence over temperature and the T m . The melting temperatures of SALD AP with and without its ligands were determined and compared in order to ascertain its thermal stability.

Results and discussion
A recombinant Alpine metagenome-derived salicylaldehyde dehydrogenase (SALD AP ) was overexpressed in its soluble form in E. coli BL21 (DE3) cells and the protein was successfully purified to homogeneity using Co 2+ -affinity and gel-filtration chromatography. The elution profile of the gelfiltration chromatogram suggests that the biological unit of SALD AP is a dimer with a protein molecular mass of 115 kDa (Fig. 2b). This finding is in good agreement with the theoretical protein molecular mass of 110 kDa. Dimerization is a structural property of class 3 aldehyde dehydrogenases such as vanillin dehydrogenase and benzaldehyde dehydrogenase, and differs from the tetrameric assembly (pair of dimers) of the native conformation of class 1 and 2 ALDHs (Rodriguez-Zavala & Weiner, 2002). A sequence-identity search in the PDB reveals that SALD AP shares 48% amino-acid identity with its closest homologue, a salicylaldehyde dehydrogenase (NahF) from P. putida G7. The structure of NahF (PDB entry 4jz6) was the only available crystal structure of a salicylaldehyde dehydrogenase in the PDB prior to our findings.
The purified 6ÂHis-SALD AP was concentrated to 12 mg ml À1 and crystals suitable for diffraction were grown. Diffraction data were collected to 1.9 Å resolution from a fragment of a crystal with an original size of about 60 Â 40 Â 10 mm. The crystals grew in the orthorhombic space group C222 1 , with unit-cell parameters a = 116.8, b = 121.7, c = 318.0 Å , and diffracted to 1.9 Å resolution. A summary of the data statistics is presented in Table 3.
The final model after refinement includes 470 amino-acid residues in polypeptide chains A, B, C and D, with one bound ligand per monomer. A summary of the refinement statistics is presented in Table 4. Additionally, the model contains one glycerol molecule and 1597 water molecules. The first observed residue is Thr5 and the last is the C-terminal Ile470 in all four chains. No His tags or NAD + /NADH were observed in the electron-density maps. Several ligands were tried for refinement, including salicylaldehyde, 2-naphthaldehyde, vanillin and pyrene-1-carboxaldehyde. The latter molecule was too large for the observed ligand density, while the former three molecules all fitted well in the electron density (ED); however, they still showed some residual positive ED in the Fourier map after refinement. Although the EDs were good, protocatechuic acid (PCA) was chosen as the ligand bound to the active site for refinement as it fitted the ED better. A hydrogen bond between the para-hydroxyl group of the ligand and the Asp427 side chain is indicated as a black broken line   in Fig. 3(a). The electron-density map of the enzyme without the ligand is shown in Fig. 3(b). Interestingly, the binding of a carboxylic acid in the active site of the aldehyde dehydrogenase indicates the potential for product inhibition of the enzyme during aldehyde oxidation; this also means that the enzyme may possess carboxylic acid reductase activity. In the crystal structure of NahF, Coitinho et al. (2016) showed the binding of salicylaldehyde to an invariant cysteine residue that is present in all ALDHs. The mechanism of binding and catalysis of aldehydes in ALDHs has been well studied; however, attention has not been paid to the role of carboxylic acids as ligands of ALDHs. Our finding opens up the possibility of studying the mechanism(s) of product inhibition and potential biocatalysis of carboxylic acids using this enzyme and other related aldehyde dehydrogenases.   Figure 3 (a) The electron density seen in the active site of independent molecule A after refinement; there are similar interactions in molecules B, C and D (not shown). The light blue chicken-wire nets are the 2F o À F c Fourier map with a cutoff of 1, while those in green are the F o À F c difference map at +3 cutoff and À3 cutoff. The ligand, PCA/DHB, is drawn with yellow C atoms and salicylaldehyde dehydrogenase residues are drawn with magenta C atoms; water molecules are shown as red spheres. (b) A representation of the difference electron-density map (F o À F c ) is shown as a green chicken-wire net after refinement of the structure without the ligands of the four independent molecules. The map was drawn at a 3.0 level at the ligand-binding site of molecule C (which shows the highest difference density peak in the difference map). The protein is shown in stick representation, while the position of the ligand in the complex structure is shown in line representation for comparison. A cutoff of 3.5 Å radius around the ligand atoms was used in drawing the difference map. To prove that PCA is a putative ligand of the enzyme, we carried out differential scanning fluorimetry (DSF) to show that the ligand stabilizes the protein upon binding. DSF shows that SALD AP is thermally stable at $68 C, which is higher than the thermostability reported for some yeast ALDHs (Datta et al., 2016(Datta et al., , 2017. Above 68 C, SALD AP starts to melt and therefore loses its three-dimensional structure, which is required for its activity. In the presence of PCA the melting temperature (T m ) increases to 69.2 C, which reveals that the binding of such a ligand further stabilizes the protein (Fig. 4). However, further studies such as inhibition and/or biocatalysis with PCA and site-directed mutagenesis need to be carried out to ascertain that PCA is a true ligand of SALD AP and its biological relevance. Because the enzyme is an NADdependent salicylaldehyde dehydrogenase, we also carried out DSF with NAD + and salicylaldehyde individually and in combination. Interestingly, neither salicylaldehyde nor NAD + exclusively stabilized SALD AP . However, significant (p < 0.05) stabilization of the protein was observed in the presence of both the cofactor and the substrate, with a T m of $70 C ( Table  5).
The overall crystal structure of SALD AP shows two independent homodimers in the asymmetric unit (Fig. 5). Polypeptide chains A and C formed the first dimer, while chains B and D formed the second homodimer. The crystallographically independent molecules B, C and D were modelled identically to molecule A. The crystallographically independent homodimers further confirm the finding from analytical gel filtration that the biological unit of SALD AP exists as a dimer.
SALD AP adopts the standard conformation of the ALDH superfamily. The monomer shows that the enzyme is a classical aldehyde dehydrogenase showing the typical / aldehyde dehydrogenase superfamily organization with three domains: catalytic, NAD + -binding and bridging domains (Fig. 6a). The biological unit (dimer) of SALD AP is formed by the oligomerization of two monomers through the bridging domain. The bridging or oligomerization domain is characterized by three -sheets (3, 4 and 18) that run antiparallel. The formation of the dimer involves interactions between -helices 11 (residues 217-231) of each subunit and -strands of the adjacent subunit (16, residues 417-420, and 18, residues 455-461) (Fig. 6b). The oligomerization domain is typical of that found in class 3 ALDHs, with the C-terminal portion of the protein pointing away from a position that favours the interaction of a dimer-dimer interface (tetramer), thus only favouring the formation of a dimer. Rodriguez-Zavala & Weiner (2002) found a striking difference in both the sequence and the structure of the C-terminal 'tail' of ALDH1 and ALDH3, and they demonstrated that the hydrophobic surface area found in this region is the primary force that drives the formation of tetramers. This hydrophobic surface area was found to increase in the tetrameric enzyme (ALDH1) compared with the dimeric ALDH3. The C-terminus of ALDHs was also found to be ultimately The melting curves of SALD AP showing changes in melting temperature upon binding of the enzyme (a) with 1.5 mM NAD + , (b) with 2 mM protocatechuic acid and (c) with 2 mM salicylaldehyde and with a combination of 1.5 mM NAD + and 2 mM salicylaldehyde. Table 5 Thermal stability of SALD AP showing its melting temperatures (T m ) upon interaction with different ligands.
The T m of SALD AP bound to ligands was measured in the presence of 2 mM ligand and 1.5 mM NAD + . All experiments were carried out with 6 mM enzyme. The values indicate the mean of triplicate measurements AE standard deviation. All results were compared with the T m of the untreated enzyme (control) for statistical significance using one-way ANOVA and Dunnett's multiple comparison post-test. * indicates a statistically significant difference (p < 0.05) between the test and the control.

Enzyme/ligand
Melting temperature (T m ) ( C) involved in the stability of the proteins. The nucleotidebinding domain conforms to the Rossmann fold consisting of five parallel -strands (5-9) connected to six -helices (6-11). Although an NAD + molecule was not found in the cofactor-binding site, the potential residues implicated in the interaction with NAD + adopted a fold quite similar to those observed in other NAD + -dependent ALDH complex structures. Structural comparison of the newly solved SALD AP structure with crystal structures available in the PDB revealed ALDHs with high structural similarity to SALD AP . The structural matches were analysed using the PDB90, which is a representative subset of PDB chains in which no two chains share more than 90% sequence identity with each other. Table 6 shows the first ten homologues of the 124 structures returned by the DALI server. The homologues are arranged according to rank, Z-score and percentage sequence identity.
It is not surprising that the best structural neighbour of SALD AP is NahF (PDB entry 4jz6), which is the only salicylaldehyde dehydrogenase crystal structure that was available in the PDB prior to our crystal structure. The two crystal The overall crystal structure of SALD AP shows two homodimers in the asymmetric unit. Chains A and C forming a dimer are coloured green and gold, respectively, while chains B and D forming the second dimer are coloured blue and magenta, respectively.

Figure 6
Different representations of the overall fold of novel SALD AP showing (a) the monomer as a cartoon model with the N-and C-termini labelled. The catalytic, cofactor-binding and bridging domains are coloured red, grey and lemon, respectively. The C and O atoms of the protocatechuic acid molecule are depicted as yellow and red spheres, respectively. (b) Topology diagram. Helices are shown as tubes, while -strands are shown as arrows; both are labelled numerically. The N-and C-termini are coloured yellow. structures were superimposed (Fig. 7). Superimposition allows structural alignment of the residues and comparison of the substrate-and cofactor-binding sites. The high Z-score (Table 6) indicates high structural similarity between the two proteins, and superimposition/alignment of the structures further ascertained this similarity: 85% of the amino-acid residues structurally aligned well, with a root-mean-square deviation (r.m.s.d.) of 1.050 Å over 2580 equivalent atoms. The high Z-score and similar functional description indicate homology with possible implications for functional conservation. SALD AP has 18 -strands while NahF has 21. Conversely, NahF has 18 -helices while SALD AP has 20. In essence, these two proteins differ from each other at the N-terminus, where SALD AP has a short N-terminal tail with only two -strands.
However, in addition to the -strands possessed by SALD AP , NahF has three -sheets at the N-terminus, making it an elongated version of SALD AP . This truncation of the N-terminus of SALD AP might have happened during evolution as the region is located on the surface and makes no contact with other protein subunits. Hence, the region might not play a significant role in the protein. This finding strengthens the conclusion that proteins are evolutionarily more related by their structures than by their sequences. In favourable cases, structural similarity can reveal evolutionary connections that are difficult to detect using sequence comparisons.
The crystal structure that we have presented here will be useful in further studying the mechanisms of ligand binding (aldehydes/carboxylic acids) and catalysis in ALDHs. Also, the strategy we have reported serves as a proof of concept for the discovery and exploitation of novel enzymes from the environment. The detailed biochemical properties of recombinant SALD AP will be published in a separate, future paper. The atomic coordinates and crystal structure of SALD AP have been deposited in the Protein Data Bank with accession code 6qhn.