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Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci

Title
Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci
Authors
Lessard I.A.D.Healy V.L.Park I.-S.Walsh C.T.
Ewha Authors
박일선
Issue Date
1999
Journal Title
Biochemistry
ISSN
0006-2960JCR Link
Citation
vol. 38, no. 42, pp. 14006 - 14022
Publisher
ACS, Washington, DC, United States
Indexed
SCI; SCIE; SCOPUS scopus
Abstract
Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala2 to capture the D-Ala1- OPO32- intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH3+) form of D-Ala at subsite 2 (K(M2) of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH2) form of D-Ala (K(M2) of 0.66 mM, k(cat) of 550 min-1) is a 17-fold better substrate compared to D-lactate (K(M) of 0.69 mM, k(cat) of 32 min-1). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative ω-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala2. The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site ω-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K(M2) for D- lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA ω- loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala2 to capture the D-Ala1-OPO32- intermediate in the ligase active site. To ensure this selectivity in catalysis. VanA largely rejects the protonated (NH3+) form of D-Ala at subsite 2 (KM2 of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH2) form of D-Ala (KM2 of 0.66 mM, kcat of 550 min-1) is a 17-fold better substrate compared to D-lactate (KM of 0.69 mM, kcat of 32 min-1). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative ω-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala2. The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site ω-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in KM2 for D-lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA ω-loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.
DOI
10.1021/bi991384c
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자연과학대학 > 생명과학전공 > Journal papers
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