Structure and ligand binding in the putative anti-microbial peptide transporter protein, YejA.

YejABEF is an ATP-binding cassette transporter that is implicated in the sensitivity of Escherichia coli to anti-microbial peptides, the best-characterized example being microcin C, a peptide-nucleotide antibiotic that targets aspartyl-tRNA synthetase. Here the structure of the extracellular solute binding protein, YejA, has been determined, revealing an oligopeptide-binding protein fold enclosing a ligand-binding pocket larger than those of other peptide-binding proteins of known structure. Prominent electron density in this cavity defines an undecapeptide sequence LGEPRYAFNFN, an observation that is confirmed by mass spectrometry. In the structure, the peptide interactions with the protein are mediated by main chain hydrogen bonds with the exception of Arg5 whose guanidinium side chain makes a set of defining polar interactions with four YejA residues. More detailed characterization of purified recombinant YejA, by a combination of ESI and MALDI-mass spectrometry as well as thermal shift assays, reveals a set of YejA complexes containing overlapping peptides 10-19 residues in length. All contain the sequence LGEPRYAFN. Curiously, these peptides correspond to residues 8-26 of the mature YejA protein, which belong to a unique N-terminal extension that distinguishes YejA from other cluster C oligopeptide binding proteins of known structure. This 35-residue extension is well-ordered and packs across the surface of the protein. The undecapeptide ligand occupies only a fraction of the enclosed pocket volume suggesting the possibility that much larger peptides or peptide conjugates could be accommodated, though thermal shift assays of YejA binding to antimicrobial peptides and peptides unrelated to LGEPRYAFNFN have not provided evidence of binding. While the physiological significance of this 'auto-binding' is not clear, the experimental data suggest that it is not an artefact of the crystallization process and that it may have a function in the sensing of periplasmic or membrane stress.

The Structure of the LysR-type Transcriptional Regulator, CysB, Bound to the Inducer, N-acetylserine.

In Escherichia coli and Salmonella typhimurium, cysteine biosynthesis requires the products of 20 or more cys genes co-ordinately regulated by CysB. Under conditions of sulphur limitation and in the presence of the inducer, N-acetylserine, CysB binds to cys promoters and activates the transcription of the downstream coding sequences. CysB is a homotetramer, comprising an N-terminal DNA binding domain (DBD) and a C-terminal effector binding domain (EBD). The crystal structure of a dimeric EBD fragment of CysB from Klebsiella aerogenes revealed a protein fold similar to that seen in Lac repressor but with a different symmetry in the dimer so that the mode of DNA binding was not apparent. To elucidate the subunit arrangement in the tetramer, we determined the crystal structure of intact CysB in complex with N-acetylserine. The tetramer has two subunit types that differ in the juxtaposition of their winged helix-turn-helix DNA binding domains with respect to the effector binding domain. In the assembly, the four EBDs form a core with the DNA binding domains arranged in pairs on the surface. N-acetylserine makes extensive polar interactions in an enclosed binding site, and its binding is accompanied by substantial conformational rearrangements of surrounding residues that are propagated to the protein surface where they appear to alter the arrangement of the DNA binding domains. The results are (i) discussed in relation to the extensive mutational data available for CysB and (ii) used to propose a structural mechanism of N-acetylserine induced CysB activation.

Peptide transport in Bacillus subtilis - structure and specificity in the extracellular solute binding proteins OppA and DppE.

Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake - the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with K d values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes.

Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex.

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.

Alanine-scanning mutagenesis of protein mannosyl-transferase from Streptomyces coelicolor reveals strong activity-stability correlation.

In Actinobacteria, protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation has an important role in cell envelope physiology. In S. coelicolor, defective Pmt leads to increased susceptibility to cell wall-targeting antibiotics, including vancomycin and ß-lactams, and resistance to phage ϕC31. The aim of this study was to gain a deeper understanding of the structure and function of S. coelicolor Pmt. Sequence alignments and structural bioinformatics were used to identify target sites for an alanine-scanning mutagenesis study. Mutant alleles were introduced into pmt-deficient S. coelicolor strains using an integrative plasmid and scored for their ability to complement phage resistance and antibiotic hypersusceptibility phenotypes. Twenty-three highly conserved Pmt residues were each substituted for alanine. Six mutant alleles failed to complement the pmt▬ strains in either assay. Mapping the six corresponding residues onto a homology model of the three-dimensional structure of Pmt, indicated that five are positioned close to the predicted catalytic DE motif. Further mutagenesis to produce more conservative substitutions at these six residues produced Pmts that invariably failed to complement the DT1025 pmt▬ strain, indicating that strict residue conservation was necessary to preserve function. Cell fractionation and Western blotting of strains with the non-complementing pmt alleles revealed undetectable levels of the enzyme in either the membrane fractions or whole cell lysates. Meanwhile for all of the strains that complemented the antibiotic hypersusceptibility and phage resistance phenotypes, Pmt was readily detected in the membrane fraction. These data indicate a tight correlation between the activity of Pmt and its stability or ability to localize to the membrane.

Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis.

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (cGMP-stimulated phosphodiesterases, adenylate cyclases, FhlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.

Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners.

YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.

The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile.

Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease.

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile.

Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA.

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP-SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.

The substrate-binding protein in bacterial ABC transporters: dissecting roles in the evolution of substrate specificity.

ATP-binding cassette (ABC) transporters, although being ubiquitous in biology, often feature a subunit that is limited primarily to bacteria and archaea. This subunit, the substrate-binding protein (SBP), is a key determinant of the substrate specificity and high affinity of ABC uptake systems in these organisms. Most prokaryotes have many SBP-dependent ABC transporters that recognize a broad range of ligands from metal ions to amino acids, sugars and peptides. Herein, we review the structure and function of a number of more unusual SBPs, including an ABC transporter involved in the transport of rare furanose forms of sugars and an SBP that has evolved to specifically recognize the bacterial cell wall-derived murein tripeptide (Mtp). Both these examples illustrate that subtle changes in binding-site architecture, including changes in side chains not directly involved in ligand co-ordination, can result in significant alteration of substrate range in novel and unpredictable ways.

Structure of components of an intercellular channel complex in sporulating Bacillus subtilis.

Following asymmetric cell division during spore formation in Bacillus subtilis, a forespore expressed membrane protein SpoIIQ, interacts across an intercellular space with a mother cell-expressed membrane protein, SpoIIIAH. Their interaction can serve as a molecular "ratchet" contributing to the migration of the mother cell membrane around that of the forespore in a phagocytosis-like process termed engulfment. Upon completion of engulfment, SpoIIQ and SpoIIIAH are integral components of a recently proposed intercellular channel allowing passage from the mother cell into the forespore of factors required for late gene expression in this compartment. Here we show that the extracellular domains of SpoIIQ and SpoIIIAH form a heterodimeric complex in solution. The crystal structure of this complex reveals that SpoIIQ has a LytM-like zinc-metalloprotease fold but with an incomplete zinc coordination sphere and no metal. SpoIIIAH has an α-helical subdomain and a protruding ß-sheet subdomain, which mediates interactions with SpoIIQ. SpoIIIAH has sequence and structural homology to EscJ, a type III secretion system protein that forms a 24-fold symmetric ring. Superposition of the structures of SpoIIIAH and EscJ reveals that the SpoIIIAH protomer overlaps with two adjacent protomers of EscJ, allowing us to generate a dodecameric SpoIIIAH ring by using structural homology. Following this superposition, the SpoIIQ chains also form a closed dodecameric ring abutting the SpoIIIAH ring, producing an assembly surrounding a 60 Å channel. The dimensions and organization of the proposed complex suggest it is a plausible model for the extracellular component of a gap junction-like intercellular channel.

Crystal and solution studies reveal that the transcriptional regulator AcnR of Corynebacterium glutamicum is regulated by citrate-Mg2+ binding to a non-canonical pocket.

Corynebacterium glutamicum is an important industrial bacterium as well as a model organism for the order Corynebacteriales, whose citric acid cycle occupies a central position in energy and precursor supply. Expression of aconitase, which isomerizes citrate into isocitrate, is controlled by several transcriptional regulators, including the dimeric aconitase repressor AcnR, assigned by sequence identity to the TetR family. We report the structures of AcnR in two crystal forms together with ligand binding experiments and in vivo studies. First, there is a citrate-Mg(2+) moiety bound in both forms, not in the canonical TetR ligand binding site but rather in a second pocket more distant from the DNA binding domain. Second, the citrate-Mg(2+) binds with a KD of 6 mM, within the range of physiological significance. Third, citrate-Mg(2+) lowers the affinity of AcnR for its target DNA in vitro. Fourth, analyses of several AcnR point mutations provide evidence for the possible involvement of the corresponding residues in ligand binding, DNA binding, and signal transfer. AcnR derivatives defective in citrate-Mg(2+) binding severely inhibit growth of C. glutamicum on citrate. Finally, the structures do have a pocket corresponding to the canonical tetracycline site, and although we have not identified a ligand that binds there, comparison of the two crystal forms suggests differences in the region of the canonical pocket that may indicate a biological significance.

Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi.

Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded ß-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-ß-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the ß-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other ß-barrel proteins.

Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites.

N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex.

Formation of functional E3 ligase complexes with UBC2 and UEV1 of Leishmania mexicana.

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.

Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila.

Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and ß chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, ß = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, ß = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.

MpaA is a murein-tripeptide-specific zinc carboxypeptidase that functions as part of a catabolic pathway for peptidoglycan-derived peptides in γ-proteobacteria.

The murein peptide amidase MpaA is a cytoplasmic enzyme that processes peptides derived from the turnover of murein. We have purified the enzyme from Escherichia coli and demonstrated that it efficiently hydrolyses the γ-D-glutamyl-diaminopimelic acid bond in the murein tripeptide (L-Ala-γ-D-Glu-meso-Dap), with Km and kcat values of 0.41±0.05 mM and 38.3±10 s-1. However, it is unable to act on the murein tetrapeptide (L-Ala-γ-D-Glu-meso-Dap-D-Ala). E. coli MpaA is a homodimer containing one bound zinc ion per chain, as judged by mass spectrometric analysis and size-exclusion chromatography. To investigate the structure of MpaA we solved the crystal structure of the orthologous protein from Vibrio harveyi to 2.17 Å (1Å=0.1 nm). Vh_MpaA, which has identical enzymatic and biophysical properties to the E. coli enzyme, has high structural similarity to eukaryotic zinc carboxypeptidases. The structure confirms that MpaA is a dimeric zinc metalloprotein. Comparison of the structure of MpaA with those of other carboxypeptidases reveals additional structure that partially occludes the substrate-binding groove, perhaps explaining the narrower substrate specificity of the enzyme compared with other zinc carboxypeptidases. In γ-proteobacteria mpaA is often located adjacent to mppA which encodes a periplasmic transporter protein previously shown to bind murein tripeptide. We demonstrate that MppA can also bind murein tetrapeptide with high affinity. The genetic coupling of these genes and their related biochemical functions suggest that MpaA amidase and MppA transporter form part of a catabolic pathway for utilization of murein-derived peptides that operates in γ-proteobacteria in addition to the established murein recycling pathways.