Structure of the mycobacterial peptidoglycan amidase Rv3717 1 Structural and biochemical analyses of Mycobacterium tuberculosis N-acetylmuramyl L-alanine amidase Rv3717 point to a role in peptidoglycan fragment recycling*

Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial Nacetylmuramyl L-alanine amidase Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared to previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded beta-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn binding rearranges active site residues, and disulfide formation promotes folding of the beta-hairpin. Although Zn is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (E200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling. INTRODUCTION Tuberculosis is among the most deadly infectious diseases worldwide. The current treatments are lengthy, taking 6-9 months to complete, while emergence of strains with resistance to all front-line drugs puts emphasis on developing new antimicrobials (1). A more detailed understanding of the physiology of Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, can help improve current therapies and lay the foundation for developing new treatments. One area of bacterial physiology that can provide therapeutic targets is the mycobacterial cell wall. Peptidoglycan forms the basis for all bacterial cell walls. A polymer constructed from disaccharide pentapeptide units, it forms a single molecule that surrounds the bacterial cell, allowing it to resist turgor pressure (2). Peptidoglycan also serves as a basis for attachment of all other cell wall-associated polysaccharides, lipids, and proteins. In mycobacteria, the resulting multilayered cell wall structure provides a highly selective barrier essential for survival (3). The http://www.jbc.org/cgi/doi/10.1074/jbc.M113.510792 The latest version is at JBC Papers in Press. Published on September 9, 2013 as Manuscript M113.510792 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Structure of the mycobacterial peptidoglycan amidase Rv3717 2 mycobacterial cell wall is also a thoroughly validated drug target. Peptidoglycan hydrolases are key enzymes in cell wall metabolism and have multiple functions in shape maintenance, cell growth, daughter cell separation, peptidoglycan maturation and fragment recycling (4). Based on their chemical specificity, peptidoglycan hydrolases are divided into three classes: i) glycosidases that cleave between the sugars, ii) peptidases that target peptide bonds, and iii) N-acetylmuramyl L-alanine amidases that can separate peptides from sugar strands. Peptidoglycan hydrolases have been comparatively well studied in model organisms, Escherichia coli and Bacillus subtilis (5,6). Recently, much attention has also been given to the peptidoglycan degradation machinery of Mtb (7,8). Peptidoglycan N-acetylmuramyl L-alanine amidases fall into two families. Amidases from the Pfam family Amidase_2, in addition to bacterial and phage amidases, include eukaryotic proteins such as fly and human peptidoglycan recognition proteins with roles in anti-bacterial immunity (9). Members of the Pfam Amidase_3 Zn-dependent amidase family are limited to bacteria and phage and include the E. coli AmiA, AmiB, AmiC and B. subtilis CwlB and CwlC enzymes. CwlB, previously called LytC, is the founding member of this family and was discovered as one of two major B. subtilis autolysins (10,11). In Mtb, a peptidoglycan amidase CwlM was discovered by sequence similarity to B. subtilis amidase CwlB and its muralytic activity was confirmed experimentally (12). Structures of one bacterial (13) and two phage (14,15) N-acetylmuramyl Lalanine amidases from the Amidase_3 family have been reported. However, the structural basis of substrate binding has not yet been investigated. Here, we report the structural and biochemical analysis of Rv3717, an Mtb peptidoglycan amidase. We found that the recombinant Rv3717 protein is active on muramyl dipeptide (MDP), a model substrate (Figure 1A), but not on polymerized peptidoglycan. Structures of two partially assembled forms of Rv3717 allowed us to establish the contributions of Zn binding and disulfide formation to assembly and catalysis. Structure of the mature Rv3717 bound to Lalanine-iso-D-glutamine dipeptide, a product of MDP hydrolysis, helped identify an additional catalytic glutamate residue (E200) that does not participate in binding to Zn ions. This productbound structure demonstrates the mode of substrate binding likely shared by the Amidase_3 family proteins. EXPERIMENTAL PROCEDURES Sequence analysis of Amidase_3 family proteins. Hidden Markov Model (HMM)-based searches of bacterial proteomes were performed with the program hmmscan, part of the HMMER 3.0 package (16). The search model for Amidase_3 was downloaded from Pfam server (17) and the predicted proteomes of E. coli K12 substrain DH10B, B. subtilis 168, M. tuberculosis H37Rv, and M. smegmatis mc 155 were obtained from NCBI (GenBank: CP000948, NC_000964, NC_000962, CP000480). Protein sequences of peptidoglycan amidases identified with hmmscan were trimmed to their catalytic domains and aligned using the hmmalign program. Protein alignment was analyzed using the Phylip package (evolution.genetics.washington.edu/phylip) to produce a maximum-likelihood tree with 100 bootstrap replicates. Graphical representation of the tree was created with the FigTree program (http://tree.bio.ed.ac.uk/software/figtree/). Cloning, and expression of Rv3717. Further sequence analysis with the SignalP web service (18) revealed that Rv3717 contained a secretion signal peptide sequence at its N-terminus. Consequently, an N-terminal truncation of Rv3717 matching the sequence of the predicted mature protein (residues 20-241) was cloned into pDEST15 vector using the Gateway system (Invitrogen). N-terminal GST-fused Rv3717 was expressed by auto-induction (19) in E. coli BL-21 Codon Plus cells. Cell pellets were harvested by centrifugation and stored at -80 °C. Cells were resuspended in buffer A (300 mM NaCl, 20 mM HEPES pH 7.5, 5% glycerol, 0.5 mM TCEP) supplemented with protease inhibitors AEBSF and E-64 (0.25 mM and 1 μM, respectively). Cells were lysed by sonication, lysates were cleared by centrifugation, and glutathione affinity chromatography was carried out at room temperature using 5 mL GST-affinity columns (GE Healthcare). After elution with 30 mM glutathione in buffer A, protein was cleaved with 0.1 mg TEV protease per liter of culture while being dialyzed against buffer B (30 mM NaCl, 20 Structure of the mycobacterial peptidoglycan amidase Rv3717 3 mM HEPES, 5% glycerol, 0.5 mM TCEP). The sample was passed through GST-affinity and anion exchange Capto-Q columns (GE Healthcare) attached in tandem to achieve complete removal of the GST tag. The flow-through fraction was oxidized by addition of one-tenth final volume of oxidizing buffer C (100 mM reduced glutathione, 10 mM oxidized glutathione, 300 mM bis-tris propane pH 9, 10% glycerol, 300 mM NaCl, 10 mM zinc acetate). The sample was filtered through a 0.2 μm syringe filter and concentrated using centrifugal filters with a 10 kDa cutoff (Amicon). Concentrated oxidized protein was applied to a Superdex-75 column mounted on an FPLC instrument and preparative size-exclusion chromatography was performed against a nonreducing buffer containing 100 mM NaCl, 20 mM HEPES pH 7.5, and 10% glycerol. MDP hydrolysis by Rv3717. Reactions included 100 mM sodium phosphate buffer pH 6.5, MDP was used at 500 μM, and Rv3717 at 5 μM. Reactions were mixed and incubated at room temperature for 40 minutes and stopped by centrifugation through a 10 kDa cutoff filter. Sample aliquots of 20 μL were mixed with 100 μL o-phthalaldehyde developing solution (1.5 mM ophthalaldehyde, 350 mM sodium borate pH 9.5, 1% v/v beta-mercaptoethanol). Following a 15minute incubation, absorbance reading at 340 nm was recorded and results were analyzed in Excel. For the EDTA-treated sample, the protein was washed with 10 mM EDTA in 100 mM sodium phosphate over the concentrator, and EDTA was removed by washing two more times with EDTAfree buffer. For the EDTA-treated, Znreconstituted protein, the enzyme was treated as above and 1 mM final concentration of zinc acetate was added to the assay buffer. For the comparison of oxidized and reduced Rv3717, the protein was pre-incubated in 100 mM sodium phosphate buffer pH 8.0 with or without 10 mM TCEP for 2 hours under nitrogen atmosphere; 1 mM final concentration TCEP was included in the assay buffer for the reduced sample. Crystallographic analysis of Rv3717. All forms of Rv3717 were crystallized in 200 mM NaCl, 100 mM TRIS pH 8.5, 25% PEG 3350 by vapor diffusion in a hanging drop format. The crystals were harvested from the drops and frozen in liquid nitrogen. Data were collected at Beamline 8.3.1 at the Advanced Light Source (20) and reduced using HKL2000 (www.hkl-xray.com). Molecular replacement using Bartonella henselae AmiB as the search model (PDB: 3NE8) was performed using Phenix (21). Model building and refinement were carried out using Phenix and Coot (22). The data collection and model refinement statistics are listed in Table 1. Molecular images were generated using Chimera (23). Mapping of secondary structure to the protein alignment was performed using ESPript (http://espript.ibcp.fr/). For the surface conservation analysis of Rv3717, we used BLAST (http://blast.ncbi.nlm.nih.gov/) to gather 100 highest scoring unique protein sequences from phylum Actinobacteria (Taxonomy ID: 201174). The sequences all had greater than 95% query coverage, yet ranged in sequence identity from 38% to 100%. They were aligned using ClastalW2 algorithm on the EBI server (http://www.ebi.ac.uk/Tools/msa/clustalw2/) with default parameters. Chimera (23) was used to map percent residue conservation scores onto the protein surface. Whole B. subtilis peptidoglycan degradation. 0.1 mg of B. subtilis peptidoglycan (SigmaAldrich) in aqueous suspension was treated by 0.01 mg either Rv3717 or mutanolysin in 50 mM sodium phosphate buffer pH 6.5 for 60 hours with shaking. Mutanolysin samples were supplemented with 1 mM magnesium chloride and amidase samples with 1 mM zinc acetate. Absorbance measurements at 595 nm were used to measure the decrease in turbidity. RESULTS As part of a comprehensive annotation of Mtb peptidoglycan hydrolases, we identified Rv3717 and Rv3915, two enzymes that belong to the Pfam family Amidase_3. The founding member of this Pfam family is B. subtilis amidase CwlB, previously LytC, the family also includes E. coli AmiA, AmiB, and AmiC proteins. The Mtb hydrolase Rv3915 has been previously identified and named CwlM by Deng and colleagues (12), while Rv3717 has not yet been investigated. Phylogenetic analysis indicated that the two mycobacterial amidases form a separate clade suggesting that their diversification occurred after divergence from E. coli and B. subtilis homologs (Figure 1B). Structure of the mycobacterial peptidoglycan amidase Rv3717 4 We purified recombinant Rv3717 protein and showed that it was able to hydrolyze MDP releasing N-acetyl muramide and L-Ala-iso-D-Gln dipeptide products (Figure 1A, 1C). Rv3717 hydrolytic activity was dependent on the presence of Zn ions retained by the enzyme during gel filtration against a Zn-free buffer. Washing the enzyme with 10 mM EDTA abolished hydrolytic activity, and this effect was reversed by the addition of excess Zn to the reaction buffer (Figure 1C). We concluded that Rv3717 is a functional Zn-dependent peptidoglycan amidase. To investigate the structure of Rv3717 and its mode of ligand binding, we determined the crystal structure of the recombinant protein. Rv3717 crystallized in presence of MDP in space group P21212 with one enzyme-ligand complex per asymmetric unit. X-ray data were collected at cryogenic temperature using synchrotron radiation (Table 1). The structure was solved with molecular replacement using Bartonella henselae AmiB (PDB: 3NE8). Rather than the MDP substrate that was used in the crystallization, the electron density clearly revealed one of the products, L-Ala-iso-DGln, bound in the active site. The Rv3717 structure contains the typical features of Amidase_3 fold: a central six-stranded beta-sheet, six surrounding alpha-helices, and a Zn atom coordinated by two histidines and one glutamate in the active site (Figure 2A). In addition, Rv3717 has a unique feature a 20-amino-acid insertion that forms a short 310-helix and a beta-hairpin linked to the core enzyme with a disulfide bond (Figure 2B). This insertion is absent in homologs from model organisms as well as the paralogous Rv3915 amidase in Mtb (Figure 2C). Because this unique disulfide-bonded insertion is positioned close to the active site, we hypothesized that disulfide oxidation could affect enzyme folding and catalysis. To address this question, we solved crystal structures of reduced Rv3717 in Zn-bound and Zn-free forms. Reduced protein crystallized under the same conditions but in the space group C2 with two protein molecules per asymmetric unit. Compared to the oxidized protein structure, the model for the reduced protein contains internal gaps due to lack of electron density. In the Zn-free form, two of the Zn-binding residues, His35 and Glu70, were disordered in the B molecule, while the whole His35 residue including backbone atoms was in the wrong conformation to coordinate Zn in the A molecule (Figure 3A, top panel). When the crystals of the reduced Rv3717 were soaked with Zn-containing cryoprotectant solution, crystallographic analysis revealed reordering of the catalytic residues and reappearance of Zn density (bottom panel in Figure 3A). The beta-hairpin and the 310-helix that comprise the Rv3717 unique insertion were disordered in both forms (Figure 3B). Thus, disulfide oxidation appears dispensable for folding of the enzyme's core and of its catalytic center. Consistent with these results, reducing the disulfide bond did not alter the efficiency of MDP hydrolysis (Figure 3C). Although mature Rv3717 was crystallized in the presence of muramyl dipeptide, the electron density envelope in the active site corresponded to the dipeptide L-Ala-iso-D-Gln (Figure 4A). No density features that could accommodate the muramic acid were present. We concluded that MDP was hydrolyzed during the crystallization process and that only the dipeptide product remained bound to the enzyme. The leaving group of the peptide was coordinated by a water molecule adjacent to a conserved amino acid, Glu200, suggesting a role for this residue in catalysis (Figure 4B). Consistent with this idea, mutating Glu200 to either alanine or glutamine abolished MDP hydrolysis (Figure 4C). As a guide to define other functional sites, we focused on the conservation of the ligand-binding surface among likely Rv3717 orthologs. We used BLASTP to collect and ClustalW2 to align 100 highest scoring Rv3717 homologous sequences from the phylum Actinobacteria (95-100% query coverage, 38-100% amino acid sequence identity). We mapped amino acid sequence conservation to the Rv3717 protein surface and discovered that residues in the extended active site that interact with the bound product are highly conserved across Rv3717 homologs (Figure 4D). Thus the mode of substrate binding is likely similar among these proteins. Since other amidases have been reported to break down whole peptidoglycan sacculi, we tested the activity of Rv3717 in a standard assay based on turbidity of a peptidoglycan solution. Compared to the mutanolysin positive control, Rv3717 did not clear B. subtilis peptidoglycan suspension (Figure 5A). This inability to process Structure of the mycobacterial peptidoglycan amidase Rv3717 5 polymerized peptidoglycan is associated with a distinct feature of the substrate-binding site. The dipeptide product binds at the head of a large blind tunnel that extends into the body of the enzyme (Figure 5B). The tunnel is long enough to accommodate 2-3 additional amino acids beyond the iso-D-Gln, but not an entire peptide cross-link. In contrast, the structures of Amidase_3 enzymes that hydrolyze polymerized peptidoglycan, including Listeria phage PSA endolysin (Figure 5C), contain equivalent Zn-bound catalytic centers within more open and accessible active sites. DISCUSSION Peptidoglycan hydrolases are central players in bacterial cell wall homeostasis. Significant advances have been made investigating peptidoglycan hydrolases of model organisms, and extension of these studies to medically important species is a priority. In this work, we identified an Mtb peptidoglycan hydrolase, Rv3717, a homolog of well-studied enzymes AmiA, AmiB, and AmiC of E. coli and CwlB of B. subtilis. We confirmed that Rv3717 is a Zn-dependent peptidoglycan amidase and have determined crystal structures of the mature enzyme in complex with a dipeptide product and of two assembly intermediates. Our analysis shows that Rv3717 is a single domain protein that is likely targeted to the pseudo-periplasmic space by the signal peptide. Once outside the cytoplasm, folding is completed by Zn binding and disulfide oxidation necessary to lock down a 20-residue insertion that forms a raised rim around the active site. The core of the enzyme shared with other Amidase_3 family proteins includes the central six-stranded betasheet, two alpha helices on the face containing the active site, and four alpha-helices on the opposing face. Conserved catalytic center includes the Zn ion that activates a bound water molecule for hydrolysis of the amide bond and three Zncoordinating residues: His35, His125, and Glu70. The Rv3717 characteristic 20-residue insertion forms a short 310-helix followed by a beta-hairpin. This structural addition is anchored to the core of the enzyme by a disulfide bond. Disulfide oxidation and folding of the insertion are not required for folding of the enzyme core or in vitro MDP hydrolysis. These observations do not rule out potential contributions of this feature of the enzyme to substrate selection and in vivo activity. The structure of Rv3717 bound to the Lalanine-iso-D-glutamine dipeptide provides previously unobserved details of substrate recognition and catalysis by this protein family. We identified an additional residue in the active site, Glu200 in Rv3717, that is positioned behind the substrate and could function as a general acid/base in the reaction. This residue is conserved in Rv3717 homologs and mutations to either alanine or glutamine block catalysis. Other residues in contact with the bound dipeptide are highly conserved among the related actinobacterial amidases, including those with as little as 38% overall sequence identity, indicating that the mode of substrate binding is likely conserved among these enzymes. Structural alignment of Rv3717 to the three reported amidases shows that the dipeptide fits the surfaces of two of them, Listeria phage PSA and Clostridium phage phicd27 endolysins, without changes in position or conformation. In the third protein, Bartonella henselae AmiB, the available structure is of the inactive protein with the active site occluded by an alpha-helix (13). This observation suggests that the position of the peptide portion of the substrate relative to the catalytic Zn ion is conserved and allows prediction of ligand interacting residues in other Amidase_3 family proteins. Unlike its Mtb paralog, Rv3915, which is active on cell wall fragments and whole peptidoglycan sacculi (12), Rv3717 activity appears limited to peptidoglycan monomers. This difference could be explained by the shape of the Rv3717 catalytic pocket. A deep tunnel next to the distal end of the bound dipeptide product is formed in part by the Rv3717 characteristic insertion. The size of the tunnel provides an elegant steric mechanism for substrate selection. Although the catalytic center is accessible, polymerized peptidoglycan cannot engage the enzyme, because the peptide cross-links cannot enter the tunnel. On the other hand, because the dipeptide product does not fill the tunnel, we speculate that substrates with longer stem peptides could be accommodated. In this case, Rv3717 substrate binding would depend on a peptidase autolysin first cleaving the peptide cross-links. This hypothesis is strengthened by the observation by Griffin et al. that Mtb tolerates transposon Structure of the mycobacterial peptidoglycan amidase Rv3717 6 insertions in the rv3717 gene, but not the rv3915 gene (24). Thus, Rv3717 enzymatic activity is not required for bacterial in vitro growth. These observations together with the fact that Rv3717 is active on peptidoglycan fragments, suggest a role for this enzyme in peptidoglycan fragment recycling. The concept of peptidoglycan recycling is well established in the Gram-negative bacteria (25), with many dedicated enzymes including AmpG, an E. coli importer of N-acetylmuramyl peptides. Since Gram-positive bacteria shed large amounts of peptidoglycan fragments into the medium, it has been assumed that they do not recycle peptidoglycan. This view, however, is currently being challenged (26). In the proposed Grampositive recycling pathway, amidase activity is required for uptake of N-acetylmuramyl peptides due to absence of an AmpG-like transporter. Since Mtb, more closely related to Gram-positive bacteria, possesses a nearly impermeable outer membrane the peptidoglycan fragments produced during cell wall turnover may well be recycled. The substrate specificity of Rv3717 defined by the tunnel that limits the length of stem peptides provides a guide for studies of the role of this enzyme in vivo. ACKNOWLEDGEMENTS We thank Dr. Ksenia V. Krasileva for assistance with phylogenetic analysis and critical reading of the manuscript. We are grateful to James Holton, George Meigs, and Jane Tanamachi for assistance with Beamline 8.3.1 at the Lawrence Berkeley National Laboratory Advanced Light Source. This work has been supported by the National Institute of Allergy and Infectious Diseases (NIAID) / National Institutes of Health (NIH) grant AI095208. 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(2011) High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog. 7, e1002251 25. Park, J. T., and Uehara, T. (2008) How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiol. Mol. Biol. Rev. 72, 211-227 26. Reith, J., and Mayer, C. (2011) Peptidoglycan turnover and recycling in Gram-positive bacteria. Appl. Microbiol. Biotechnol. 92, 1-11 Structure of the mycobacterial peptidoglycan amidase Rv3717 8 FIGURE LEGENDS Figure 1. Rv3717 is a functional Zn-dependent mycobacterial N-acetylmuramyl L-Alanine amidase. A) Amidase-catalyzed hydrolysis of muramyl dipeptide produces N-acetylmuramic acid and an Lalanine-iso-D-glutamine dipeptide. B) Phylogenetic relationships among peptidoglycan amidases of E. coli, B. subtilis, M. tuberculosis, M. smegmatis, and a distantly related Listeria monocytogenes bacteriophage PSA endolysin, PlyPSA. Maximum-likelihood tree of Amidase_3 family peptidoglycan amidases. Bootstrapping values at nodes show high confidence in the mycobacterial (dark grey) and E. coli (medium grey) clades. C) Muramyl dipeptide hydrolysis by Rv3717 depends on the presence of Zn. Increase in primary amine concentration following 40-minute degradation by metal-bound and metal-free forms of Rv3717 amidase was measured using ortho-phthalaldehyde detection and corresponds to production of the dipeptide product. The error bars represent one standard deviation above the mean of three replicate measurements. Figure 2. The structure of Rv3717 is an elaboration on the Amidase_3 fold. A) Ribbon diagram of Rv3717 in complex with dipeptide L-Ala-iso-D-Gln, one of two products formed during muramyl dipeptide hydrolysis. The amidase catalytic center contains a Zn cation that is coordinated by His35, His125, and Glu70. B) Comparison of Rv3717 structure (yellow) to another Amidase_3 protein, CwlV from Paenibacillus polymyxa (PDB: 1JWQ, blue) highlights the intra-domain insertion of a short 310helix and a beta-hairpin characteristic of Rv3717 and its actinomycete orthologs. The added features are held close to the enzyme core by disulfide-bonded cysteine residues (spheres). C) Section of the protein sequence alignment highlighting the amino acid insertion (residues 41-60 in the alignment) responsible for this Rv3717 structural feature. Figure 3. Zn-binding and disulfide oxidation complete Rv3717 folding. A) In the absence of metal, the catalytic-center residues of reduced Rv3717 are in the wrong conformation to coordinate Zn (top panel), and they are reoriented following addition of the metal (bottom panel). B) The characteristic insertion that is folded in oxidized Rv3717 (yellow) is disordered in both metal-free (purple) and Znbound (salmon) structures of the reduced protein, leaving the folded core of a six-stranded beta-sheet and six alpha-helices. C) Disulfide reduction in Rv3717 does not slow MDP hydrolysis. Figure 4. L-Ala-iso-D-Gln dipeptide product bound to the Rv3717 active site identifies catalytic and substrate-binding residues. A) Electron density envelope for the dipeptide product, but not the full MDP or N-acetyl muramic acid, is present in the active site. B) Residues involved in catalysis include the Znbinding triad of His35, Glu70, and His125 along with the conserved Glu200. C) Glu200 is required for substrate hydrolysis. D) Surface of Rv3717 amidase colored by residue conservation among 100 actinobacterial homologs highlights the conserved extended substrate-binding surface around the active site. Figure 5. Rv3717 amidase activity is restricted to peptidoglycan fragments. A) B. subtilis peptidoglycan sacculi are solubilized by mutanolysin but not Rv3717, detected by the drop in optical density. B) Rv3717 substrate binding site extends towards a closed tunnel that could limit substrate selection. Section through the Rv3717 active site shows the bound dipeptide extending away from the catalytic center towards a hydrophobic well in the enzyme surface. Hydrophobic surfaces are shown in orange, hydrophilic in blue. C) The same section through the catalytic domain of a homologous phage amidase (Listeria phage PSA endolysin, PDB: 1XOV) shows a substrate-binding site that is more solvent exposed. Structure of the mycobacterial peptidoglycan amidase Rv3717 9 Table 1. Data collection and refinement statistics. Rv3717 L-Ala-iso-D-Gln complex Rv3717 reduced, metal-free form Rv3717 reduced, Zn-bound form Data Collection Wavelength (Å) 1.12 1.12 1.28 Temperature (K) 100 100 100 Space group P 21212 C2 C2 Cell parameters a b c (Å) 56.0/76.0/49.5 125.4/45.7/67.8 125.4/45.9/68.1 α β γ () 90/90/90 90/116.0/90 90/116.3/90 Asymmetric unit copies 1 2 2 Resolution (Å) a 50.0 – 2.10 (2.14 – 2.10) 50.0 – 2.20 (2.24 –2.20) 50.0 – 2.67 (2.72 – 2.67) Rsym (%) 15.8 (52.7) 9.1 (51.9) 14.4 (77.7) I/σI 8.65 (3.27) 9.8 (1.4) 13.1 (1.5) Completeness (%) 97.54 (87.31) 97.13 (94.67) 95.99 (82.89) Redundancy 6.8 (5.5) 2.3 (2.3) 7.5 (5.1) Refinement Resolution (Å) 45.07 – 2.10 42.33 – 2.19 42.52 – 2.67 Number of reflections 12,431 16,314 9,721 Rwork/Rfree (%) 20.63/24.46 20.46/23.28 19.7/22.32 Number of atoms Protein 1565 2599 2660 Solvent 66 79 18 B factors Protein (Å) 32.3 37.5 35.6 Solvent (Å) 33.4 36.6 22.6 Root mean square deviations Bond lengths (Å) 0.008 0.005 0.005 Bond angles (°) 0.850 1.186 1.102 Ramachandran plot Favored (%) 97 98 98 Disallowed (%) 0 0 0 PDB ID 4M6G 4M6H 4M6I a Values in parentheses refer to the highest resolution shell. Structure of the mycobacterial peptidoglycan amidase Rv3717