Gene vanXYC encodes D,D -dipeptidase (VanX) and D,D-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174

VanX and VanY have strict D,D-dipeptidase and D,D-carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala) in glycopeptide-resistant enterococci in which the C-terminal D-Ala residue has been replaced by D-lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in D-Ala-D-serine (D-Ala-D-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catalyses synthesis of D-Ala-D-Ser has a polar effect on both D, D-dipeptidase and D,D-carboxypeptidase activities. The open reading frame downstream from vanC-1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX- and VanY-type enzymes. It had very low dipeptidase activity against D-Ala-D-Ser, unlike VanX, and no activity against UDP-MurNAc-pentapeptide[D-Ser], unlike VanY. The introduction of plasmid pAT708(vanC-1,XYC) or pAT717(vanXYC) into vancomycin-susceptible Enterococcus faecalis JH2-2 conferred low-level vancomycin resistance only when D-Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2-2 and JH2-2(pAT708) and JH2-2(pAT717) indicated that the function of VanXYC was hydrolysis of D-Ala-D-Ala and removal of D-Ala from UDP-MurNAc-pentapeptide[D-Ala]. VanC-1 and VanXYC were essential, but not sufficient, for vancomycin resistance.     

Purification and characterization of VanXY(C), a D,D-dipeptidase/D,D-carboxypeptidase in vancomycin-resistant Enterococcus gallinarum BM4174.

VanXY(C), a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His(6)-VanXY(C) were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K(m) was 3.6 mm and k(cat), 2.5 s(-1); for UDP-MurNAc-L-Ala-D-Glu-L-Lys-DAla-D-Ala (UDP-MurNAc-pentapeptide[Ala]), K(m) was 18.8 mm and k(cat) 6.2 s(-1); for D-Ala-D-Ser, K(m) was 15.5 mm and k(cat) 0.35 s(-1). His(6)-VanXYC was inactive against the peptidoglycan precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNAc-pentapeptide[Ser]). The rate of hydrolysis of the terminal D-Ala of UDP-MurNAc-pentapeptide[Ala] was inhibited 30% by 2 mm D-Ala-D-Ser or UDP-MurNAc-pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in D-Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in D-Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan.Mutation of an aspartate residue (Asp59) of His-tagged VanXY(C) corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (k(cat)/K(m)) for D-Ala-D-Ala. This situation is in contrast to VanX in which mutation of Asp68-->Ala produced a greater than 200,000-fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.     

Glycopeptide resistance mediated by enterococcal transposon Tn 1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine

Cloning and nucleotide sequencing indicated that transposon Tn 1546 from Enterococcus faecium BM4147 encodes a 23365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2-2 derivative producing VanH, VanA and VanX accumulated mainly UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Lac (pentadepsipeptide) and small amounts of UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D-dipeptidase that hydrolysed D-Ala-D-Ala. Pentadepsipeptide, pentapeptide and D-Ala-D-Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D-dipeptidase that hydrolyses D-Ala-D-Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D-Ala-D-Ala-containing peptidoglycan precursors at the cell surface.     

Characterization of VanY(n) , a novel d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance in Nonomuraea sp. ATCC 39727

VanY(n) is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC?39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase?III of clinical development. VanY(n) (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanY(n) was successfully expressed as a soluble and active protein in Escherichia?coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn(2+) and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanY(n) as in VanY, VanX and VanXY Zn(2+) -dependent d,d-carboxypeptidases and d-Ala-d-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanY(n) shows d,d-carboxypeptidase and d,d-dipeptidase activity, but lacks d,d-carboxyesterase ability on d-Ala-d-Lac-terminating peptides. VanY(n) belongs to the metallo-d,d-carboxypeptidase family, but it is inhibited by β-lactams. Its characterization provides new insights into the evolution and transfer of resistance determinants from environmental glycopeptide-producing actinomycetes (such as Nonomuraea sp.) to glycopeptide-resistant pathogens (enterococci and staphylococci). It may also contribute to an early warning system for emerging resistance mechanisms following the introduction into clinics of a second-generation glycopeptide such as dalbavancin. Database The nucleotide sequence of vanY(n) is available in the GenBank data base under accession number CAD91202.     

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica

The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.     

Peptidoglycan synthesis and structure in Staphylococcus haemolyticus expressing increasing levels of resistance to glycopeptide antibiotics.

The structures of cytoplasmic peptidoglycan precursor and mature peptidoglycan of an isogenic series of Staphylococcus haemolyticus strains expressing increasing levels of resistance to the glycopeptide antibiotics teicoplanin and vancomycin (MICs, 8 to 32 and 4 to 16 microg/ml, respectively) were determined. High-performance liquid chromatography, mass spectrometry, amino acid analysis, digestion by R39 D,D-carboxypeptidase, and N-terminal amino acid sequencing were utilized. UDP-muramyl-tetrapeptide-D-lactate constituted 1.7% of total cytoplasmic peptidoglycan precursors in the most resistant strain. It is not clear if this amount of depsipeptide precursor can account for the levels of resistance achieved by this strain. Detailed structural analysis of mature peptidoglycan, examined for the first time for this species, revealed that the peptidoglycan of these strains, like that of other staphylococci, is highly cross-linked and is composed of a lysine muropeptide acceptor containing a substitution at its epsilon-amino position of a glycine-containing cross bridge to the D-Ala 4 of the donor, with disaccharide-pentapeptide frequently serving as an acceptor for transpeptidation. The predominant cross bridges were found to be COOH-Gly-Gly-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Gly-Gly-NH2. Liquid chromatography-mass spectrometry analysis of the peptidoglycan of resistant strains revealed polymeric muropeptides bearing cross bridges containing an additional serine in place of glycine (probable structures, COOH-Gly-Ser-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Ser-Gly-NH2). Muropeptides bearing an additional serine in their cross bridges are estimated to account for 13.6% of peptidoglycan analyzed from resistant strains of S. haemolyticus. A soluble glycopeptide target (L-Ala-gamma-D-iso-glutamyl-L-Lys-D-Ala-D-Ala) was able to more effectively compete for vancomycin when assayed in the presence of resistant cells than when assayed in the presence of susceptible cells, suggesting that some of the resistance was directed towards the cooperativity of glycopeptide binding to its target. These results are consistent with a hypothesis that alterations at the level of the cross bridge might interfere with the binding of glycopeptide dimers and therefore with the cooperative binding of the antibiotic to its target in situ. Glycopeptide resistance in S. haemolyticus may be multifactorial.     

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.     

Evolution of peptidoglycan biosynthesis under the selective pressure of antibiotics in Gram-positive bacteria

Acquisition of resistance to the two classes of antibiotics therapeutically used against Gram-positive bacteria, the glycopeptides and the beta-lactams, has revealed an unexpected flexibility in the peptidoglycan assembly pathway. Glycopeptides select for diversification of the fifth position of stem pentapeptides because replacement of D-Ala by D-lactate or D-Ser at this position prevents binding of the drugs to peptidoglycan precursors. The substitution is generally well tolerated by the classical D,D-transpeptidases belonging to the penicillin-binding protein family, except by low-affinity enzymes. Total elimination of the fifth residue by a D,D-carboxypeptidase requires a novel cross-linking enzyme able to process the resulting tetrapeptide stems. This enzyme, an L,D-transpeptidase, confers cross-resistance to beta-lactams and glycopeptides. Diversification of the side chain of the precursors, presumably in response to the selective pressure of peptidoglycan endopeptidases, is controlled by aminoacyl transferases of the Fem family that redirect specific aminoacyl-tRNAs from translation to peptidoglycan synthesis. Diversification of the side chains has been accompanied by a parallel divergent evolution of the substrate specificity of the L,D-transpeptidases, in contrast to the D,D-transpeptidases, which display an unexpected broad specificity. This review focuses on the role of antibiotics in selecting or counter-selecting diversification of the structure of peptidoglycan precursors and their mode of polymerization.     

Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.     

Sequence of the vanY gene required for production of a vancomycin-inducible D,D-carboxypeptidase in Enterococcus faecium BM4147.

Cloning and nucleotide sequencing identified the vanY gene as a member of the vancomycin-resistance van gene cluster of enterococcal plasmid, pIP816. The vanY gene was necessary for synthesis of the vancomycin-inducible D,D-carboxypeptidase activity previously proposed to be responsible for glycopeptide resistance. However, this activity was not required for peptidoglycan synthesis in the presence of glycopeptides. The deduced product of vanY did not display significant similarity with other D,D-carboxypeptidases.     

Mechanism of action of oritavancin and related glycopeptide antibiotics

Oritavancin (LY333328) is a semisynthetic glycopeptide antibiotic having excellent bactericidal activity against glycopeptide-susceptible and -resistant Gram-positive bacteria. Oritavancin is the N-alkyl-p-chlorophenylbenzyl derivative of chloroeremomycin (LY264826) and is currently in phase III clinical trials for use in Gram-positive infections. Studies show that oritavancin and related alkyl glycopeptides inhibit bacterial cell wall formation by blocking the transglycosylation step in peptidoglycan biosynthesis in a substrate-dependent manner. As with other glycopeptide antibiotics, including vancomycin, the effects of oritavancin on cell wall synthesis are attributable to interactions with dipeptidyl residues of peptidoglycan precursors. Unlike vancomycin, however, oritavancin is strongly dimerized and can anchor to the cytoplasmic membrane, the latter facilitated by its alkyl side chain. Cooperative interactions derived from dimerization and membrane anchoring in situ can be of sufficient strength to enable binding to either dipeptidyl or didepsipeptidyl peptidoglycan residues of vancomycin-susceptible and -resistant enterococci, respectively. This review describes the antibacterial activity of oritavancin, and examines the evidence supporting the proposed mechanism of action for this agent and related analogs.     

Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance

As one of key bacterial proteins involved in vancomycin resistance, VanX is a D,D-dipeptidase that impedes bacterial cell wall biosynthesis by hydrolyzing the essential D-Ala-D-Ala dipeptide. Based on a report by Crowder and co-workers that L-alanine-p-nitroanilide (L-Ala-pNA) was a useful substrate for continuous assay of VanX, we constructed a library of 35 L- and D-amino acid p-nitroanilides to provide the needed diversity to discover new substrates that are more specific than L-Ala-pNA. We report here that, among all compounds tested, D-leucine-p-nitroanilide (D-Leu-pNA) was found to be the best substrate for VanX enzyme (KM=8.9+/-1.2 mM, kcat=0.0102+/-0.0016 s(-1), kcat/KM=0.0012 mM(-1)s(-1)). Although it is catalytically inefficient, this new VanX substrate needs essentially no sophisticated synthetic chemistry for preparation and therefore offers a convenient means for routine analysis of enzyme catalysis and the screening of potential inhibitors. Moreover, because it is the uncommon leucine in its D form in D-Leu-pNA, enzymatic activities due to other contaminated species in Escherichia coli used for VanX overproduction should be greatly reduced.     

Selectivity for D-lactate incorporation into the peptidoglycan precursors of Lactobacillus plantarum: role of Aad, a VanX-like D-alanyl-D-alanine dipeptidase

Lactobacillus plantarum produces peptidoglycan precursors ending in D-lactate instead of D-alanine, making the bacterium intrinsically resistant to vancomycin. The ligase Ddl of L. plantarum plays a central role in this specificity by synthesizing D-alanyl-D-lactate depsipeptides that are added to the precursor peptide chain by the enzyme MurF. Here we show that L. plantarum also encodes a D-Ala-D-Ala dipeptidase, Aad, which eliminates D-alanyl-D-alanine dipeptides that are produced by the Ddl ligase, thereby preventing their incorporation into the precursors. Although D-alanine-ended precursors can be incorporated into the cell wall, inactivation of Aad failed to suppress growth defects of L. plantarum mutants deficient in d-lactate-ended precursor synthesis.     

Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci.

Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala(2) to capture the D-Ala(1)-OPO(3)(2)(-) intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH(3)(+)) form of D-Ala at subsite 2 (K(M2) of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH(2)) form of D-Ala (K(M2) of 0.66 mM, k(cat) of 550 min(-)(1)) is a 17-fold better substrate compared to D-lactate (K(M) of 0.69 mM, k(cat) of 32 min(-)(1)). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative omega-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala(2). The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site omega-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K(M2) for D-lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA omega-loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.     

Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.     

Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori

D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.