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Crystallization and preliminary X-ray analysis of a D-Ala:D-Ser ligase associated with VanG-type vancomycin resistance.

ABSTRACT: Acquired VanG-type resistance to vancomycin in Enterococcus faecalis BM4518 arises from inducible synthesis of peptidoglycan precursors ending in D-alanyl-D-serine, to which vancomycin exhibits low binding affinity. VanG, a D-alanine:D-serine ligase, catalyzes the ATP-dependent synthesis of the D-Ala-D-Ser dipeptide, which is incorporated into the peptidoglycan synthesis of VanG-type vancomycin-resistant strains. Here, the purification, crystallization and preliminary crystallographic analysis of VanG in complex with ADP are reported. The crystal belonged to space group P3(1)21, with unit-cell parameters a = b = 116.1, c = 177.2 A, and contained two molecules in the asymmetric unit. A complete data set has been collected to 2.35 A resolution from a single crystal under cryogenic conditions using synchrotron radiation.

SUBMITTER: Weber P

PROVIDER: S-EPMC2765892 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray analysis of a D-Ala:D-Ser ligase associated with VanG-type vancomycin resistance.

Weber Patrick P Meziane-Cherif Djalal D Haouz Ahmed A Saul Frederick A FA Courvalin Patrice P

Acta crystallographica. Section F, Structural biology and crystallization communications 20090923 Pt 10

Acquired VanG-type resistance to vancomycin in Enterococcus faecalis BM4518 arises from inducible synthesis of peptidoglycan precursors ending in D-alanyl-D-serine, to which vancomycin exhibits low binding affinity. VanG, a D-alanine:D-serine ligase, catalyzes the ATP-dependent synthesis of the D-Ala-D-Ser dipeptide, which is incorporated into the peptidoglycan synthesis of VanG-type vancomycin-resistant strains. Here, the purification, crystallization and preliminary crystallographic analysis o ...[more]

PMID: 19851013

Similar Datasets

Structural and functional characterization of VanG D-Ala:D-Ser ligase associated with vancomycin resistance in Enterococcus faecalis.

Project description:d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.

| S-EPMC3488035 | biostudies-literature

D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium.

Project description:Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.

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Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase.

Project description:The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid pentapeptide intermediate terminating in D-Ala-D-Ser instead of D-Ala-D-Ala, it has been predicted that the VanC ligase would be a D-Ala-D-Ser rather than a D-Ala-D-Ala ligase. Overproduction of the E. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent D-Ala-D-Ser ligase activity. The kcat/Km2 (Km2 = Km for D-Ser or C-terminal D-Ala) ratio for D-Ala-D-Ser/D-Ala-D-Ala dipeptide formation is 270/0.69 for a 400-fold selection against D-Ala in the C-terminal position. VanC2 also has substantial D-Ala-D-Asn ligase activity (kcat/Km2 = 74 mM-1min-1).

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VanG-type vancomycin-resistant Enterococcus faecalis strains isolated in Canada.

Project description:Enterococcus faecalis G1-0247 (vancomycin MIC, 16 microg/ml) was found to harbor a vanG operon 99% identical to the vanG operon in E. faecalis BM4518. E. faecalis N03-0233 (vancomycin MIC, 16 microg/ml) was found to harbor a novel vanG operon, vanG2, on an element in a different chromosomal location than the vanG-harboring elements in G1-0247 and BM4518.

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Project description:4-Coumarate:CoA ligase 2 (4CL2) from Arabidopsis thaliana catalyzes the ATP-dependent formation of the 4-coumaroyl-CoA thioester through the formation of 4-coumarate-AMP. Recombinant 4CL2 protein was expressed in Escherichia coli and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a=91.6, b=55.5, c=124.4?Å, ?=?=90.0, ?=111.1°.

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Vancomycin resistance: modeling backbone variants with D-Ala-D-Ala and D-Ala-D-Lac peptides.

Project description:To seek vancomycin analogs with broader antibacterial activity, effects of backbone modifications for the agylcon 2 on binding with D-Ala-D-Ala- and D-Ala-D-Lac-containing peptides were investigated by Monte Carlo/free energy perturbation (MC/FEP) calculations. The experimental trend in binding affinities for 2 with three tripeptides was well reproduced. Possible modifications of the peptide bond between residues 4 and 5 were then considered, specifically for conversion of the OCNH linkage to CH(2)NH(2)(+) (6), FCCH (7), HCCH (8), and HNCO (9). The MC/FEP results did not yield binding improvements for 7, 8, and 9, though the fluorovinyl replacement is relatively benign. The previously reported analog 6 remains as the only variant that exhibits improved affinity for the D-Ala-D-Lac sequence and acceptable affinity for the D-Ala-D-Ala sequence.

| S-EPMC2702131 | biostudies-literature

Genetic characterization of vanG, a novel vancomycin resistance locus of Enterococcus faecalis.

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D-Ala:D-Ala ligase gene flanking the vanC cluster: evidence for presence of three ligase genes in vancomycin-resistant Enterococcus gallinarum BM4174.

Project description:An open reading frame located 230 nucleotides downstream from the stop codon of vanS(c) and in the opposite direction relative to the other genes of the vanC cluster was identified in Enterococcus gallinarum BM4174. This gene (designated ddl2) encoded a protein of 343 amino acids that had significant predicted structural similarity to D-Ala:D-Ala ligases and displayed 33 and 35% amino acid identity to VanC-1 and the previously reported partial sequence of Ddl from E. gallinarum, respectively. Biochemical characterization by thin-layer chromatography confirmed that Ddl2 is a D-Ala:D-Ala ligase with no detectable D-Ala:D-Ser ligase activity. The vancomycin dependence of Enterococcus faecalis BM4320 (ddl mutant) was lost on electroporation of a plasmid construct expressing ddl2 constitutively. The latter strain was able to grow in the absence of vancomycin, and peptidoglycan precursor analysis under the same conditions indicated the synthesis of pentapeptide[D-Ala] as the main precursor, confirming the activity of Ddl2 in vivo. Expression of ddl and ddl2 in BM4174 was tested by reverse transcription-PCR: results suggested that both D-Ala:D-Ala ligases were expressed concomitantly. Our findings indicate that vancomycin-resistant E. gallinarum BM4174 is likely to express one D-Ala:D-Ser and two D-Ala:D-Ala ligase genes.

| S-EPMC126995 | biostudies-literature

Molecular characterization of Enterococcus faecalis N06-0364 with low-level vancomycin resistance harboring a novel D-Ala-D-Ser gene cluster, vanL.

Project description:Enterococcus faecalis N06-0364, exhibiting a vancomycin MIC of 8 microg/ml, was found to harbor a novel D-Ala-D-Ser gene cluster, designated vanL. The vanL gene cluster was similar in organization to the vanC operon, but the VanT serine racemase was encoded by two separate genes, vanTm(L) (membrane binding) and vanTr(L) (racemase).

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Crystallization and preliminary X-ray diffraction analysis of biotin acetyl-CoA carboxylase ligase (BirA) from Mycobacterium tuberculosis.

Project description:The gene encoding biotin acetyl-CoA carboxylase ligase (BirA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli with a C-terminal Strep-tag. PEG 4000 as well as PEG 8000 were used as precipitants at pH 7.5 to crystallize the protein using the vapour-diffusion technique. X-ray characterization of crystals at room temperature indicated that the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 79.7, b = 62.8, c = 105.8 A. Assuming the presence of two BirA molecules in the asymmetric unit, the solvent content of the crystals was 44% (V(M) = 2.2 A(3) Da(-1)). When transferred to a cryoprotectant, crystals grown in the same drop exhibited a difference in one unit-cell parameter, with a = 60.1, b = 64.0, c = 103.6 A, but belonged to the same P2(1)2(1)2(1) space group. These crystals, with two molecules of BirA present per asymmetric unit, appeared to have a very low solvent content of 28% (V(M) = 1.7 A(3) Da(-1)).

| S-EPMC2496853 | biostudies-literature

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