Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA.
Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
T D
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Bugg
G D
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Wright
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Dutka-Malen
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Arthur
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Courvalin
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Walsh
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019729:Carbon-Oxygen Ligases; D004352:Drug Resistance, Microbial / Q000235:genetics; D016984:Enterococcus faecium / Q000187:drug effects* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000096:biosynthesis; D016415:Sequence Alignment; D013379:Substrate Specificity; D014640:Vancomycin / Q000378:metabolism / Q000494:pharmacology*
D001426:Bacterial Proteins; D011498:Protein Precursors; C071437:VanA ligase, Bacteria; C070700:VanH protein, Enterococcus faecium; D014640:Vancomycin; D007770:L-Lactate Dehydrogenase; D019729:Carbon-Oxygen Ligases