Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA

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.

Structures of Staphylococcus aureus Cell-Wall Complexes with Vancomycin, Eremomycin, and Chloroeremomycin Derivatives by 13C{19F} and 15N{19F} Rotational-Echo Double Resonance

“…aureus cells were grown in 300 ml of defined media (20) containing [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glycine and L-[ε -15 N]lysine. The cells were harvested at OD 660 = 1.0 and were complexed with 6.7 mg (4.0 μmoles) of FPBV, resulting in 66% cell-wall binding-site occupancy (15).…”

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 VanA phenotype has been best studied and found to require the expression of five genes where all five encoded proteins VanR, -S, -H, -A, -X have enzymatic activities (2,4). VanS and -R act as partners in a two-component regulatory system, VanS as a transmembrane sensor kinase (5,6) and VanR as a response regulator (6,7), which account for inducible transcriptional activation of VanH, -A, and -X. VanH encodes a D-Lac dehydrogenase, functioning as a D-specific pyruvate reductase (8) (8).…”

“…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).…”

Christopher Walsh: Pioneer and innovator in antibiotic and natural product chemical biology

“…3 This project has its roots in Walsh's earlier exploration of the mechanism of inhibition of alanine racemase by b-fluroalanine in late 1970s, the enzyme that generates D-alanine essential for bacterial cell wall synthesis and vancomycin activity. At the same time, his interests in the biosynthesis of the siderophore enterobactin were gaining momentum.…”