Identification of vancomycin resistance protein VanA as a D-alanine:D-alanine ligase of altered substrate specificity

Abstract

High-level glycopeptide resistance in Enterococcus faecium BM4147 is mediated by a 38-kDa protein VanA, whose amino acid sequence is related to Gram-negative D-alanine:D-alanine (D-Ala-D-Ala) ligases [Dutka-Malen, S., Molinas, C., Arthur, M., & Courvalin, P. (1990) Mol. Gen. Genet. 224, 364-372]. We report purification of VanA and demonstrate that it has D-Ala-D-Ala ligase activity but has substantially modified substrate specificity, compared with Gram-negative D-Ala-D-Ala ligases. VanA preferentially condenses D-Ala with D-Met or D-Phe, raising the possibility that its cellular role is to synthesize a modified cell-wall component, which is subsequently not recognized by vancomycin.

D-Alanyl-D-Lactate and D-Alanyl-D-Alanine Synthesis by D-Alanyl-D-Alanine Ligase from Vancomycin-resistant Leuconostoc mesenteroides EFFECTS OF A PHENYLALANINE 261 TO TYROSINE MUTATION

“…The molecular analysis of vancomycin resistance has led to the similarities and differences between the dipeptide forming D-Ala-D-Ala ligases and the ϳ28% identical 38 kDa D-Ala-D-Ala ligase homolog VanA, whose gain of depsipeptide ligase activity is crucial for phenotypic resistance (8,9). The x-ray structure of the DdlB isoform of Escherichia coli in complex with a phosphinophosphate analog of a dipeptidyl reaction intermediate has allowed definition of the ligase active site (11) and predicted functions for several residues that were validated by mutagenesis (12).…”

“…Enzyme Assay by TLC-The assay mixture included 0.2 mM D-[1-14 C]-Ala (0.1 mCi/ml, 55 Ci/ mol) or 0.2 mM D-[1-14 C]-Lac (0.1 mCi/ml, 55 Ci/ mol), 100 mM HEPES, pH 7.5, 10 mM KCl, 10 mM MgCl 2 , 10 mM ATP, and additional unlabeled D-Ala, D-Lac, or DL-Hbut with enzyme (ϳ10 M), which was incubated at room temperature for 3 h. Three l of each sample was analyzed on TLC cellulose plate as described previously (8,9).…”

Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583

“…Stepwise elutions at 100 mM and 300 mM NaCl from Q-Sepharose allowed vanB protein recovery in the high salt eluent. Unlike VanA [5], VanB was found to aggregate upon ammonium sulfate fractionation, suggesting a difference in hydrophobic surface pattern. ATP-agarose chromatography was finally used to eliminate contaminating ATPase activity which interfered with the n-Ala:p-Ala ligase ADP release coupled assay.…”

“…Enzyme activity was monitored by thin-layer chromatographic assay [5] or, when possible, by the ADP release coupled assay [7'J Protein concentration was determined by measurement of the absorbance at 280 nm.…”

“…Isolates with high-level resistance to vancomycin (MIC from 64 to l,OOO,&ml) and to teicoplanin (MIC from 16 to 512 ,@ml) belong to the Van4 phenotype. This inducible resistance is associated with production of the VanA protein, a D-Ala: D-Ala ligase of altered substrate specificity which preferentially catalyzes ester bond formation between D-Ala and a D-Zhydroxyacid [5,6]. D-Lactate (D-Lac) was identified as the in vivo substrate for VanA [2,10].…”