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PMID:1931965
| Citation |
Bugg, TD, Wright, GD, Dutka-Malen, S, Arthur, M, Courvalin, P and Walsh, CT (1991) Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry 30:10408-15 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Carbon-Oxygen Ligases; Drug Resistance, Microbial/genetics; Enterococcus faecium/drug effects; Enterococcus faecium/genetics; Genes, Bacterial; Kinetics; L-Lactate Dehydrogenase/metabolism; Molecular Sequence Data; Protein Precursors/biosynthesis; Sequence Alignment; Substrate Specificity; Vancomycin/metabolism; Vancomycin/pharmacology |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
|
enables |
GO:0008716: D-alanine-D-alanine ligase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
| GO:0008716: D-alanine-D-alanine ligase activity |
ECO:0000314: |
F |
Fig. 2 is a TLC showing the D-alanine-D-alanine ligase activity of VanA. In the presence of VanA, the TLC shows that [14C]D-alanine is converted to [14C]D-alanine-D-alanine. In the absence of VanA, [14C]D-alanine does not form [14C]D-alanine-D-alanine. |
complete | ||||
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