Molecular mechanism of VanHst, an alpha-ketoacid dehydrogenase required for glycopeptide antibiotic resistance from a glycopeptide producing organism.
The vancomycin resistance enzyme VanH is an alpha-ketoacid dehydrogenase that stereospecifically reduces pyruvate to D-lactate, which is required for the synthesis of the depsipeptide D-alanine-D-lactate. This compound then forms an integral part of the bacterial cell wall replacing the vancomycin target dipeptide D-alanine-D-alanine, thus the presence of VanH is essential for glycopeptide resistance. In this work, the VanH homologue from the glycopeptide antibiotic producing organism Streptomyces toyocaensis NRRL 15009, VanHst, has been overexpressed in Escherichia coli and purified, and its substrate specificity and mechanism were probed by steady-state kinetic methods and site-directed mutagenesis. The enzyme is highly efficient at pyruvate reduction with kcat/Km = 1.3 x 10(5) M-1 s-1 and has a more restricted alpha-ketoacid substrate specificity than VanH from vancomycin resistant enterococci (VRE). Conversely, VanHst shows no preference between NADH and NADPH while VanH from VRE prefers NADPH. The kinetic mechanism for VanHst was determined using product and dead-end inhibitors to be ordered BiBi with NADH binding first followed by pyruvate and products leaving in the order D-lactate, NAD+. Site-directed mutagenesis indicated that Arg237 plays a role in pyruvate binding and catalysis and that His298 is a candidate for an active-site proton donor. Glu266, which has been suggested to modulate the pKa of the catalytic His in other D-lactate dehydrogenases, was found to fulfill a similar role in VanHst, lowering a pKa value of kcat/Km nearly 2 units. These results now provide the framework for additional structure and inhibitor design work on the VanH family of antibiotic resistance enzymes.
Marshall
C G
CG
Antimicrobial Research Centre, Department of Biochemistry, McMaster University, Hamilton, ON, Canada.
Zolli
M
M
Wright
G D
GD
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001667:Binding, Competitive; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006020:Glycopeptides / Q000096:biosynthesis*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D045022:Lactate Dehydrogenases; D016297:Mutagenesis, Site-Directed; D010072:Oxamic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013237:Stereoisomerism; D013302:Streptomyces / Q000187:drug effects / Q000201:enzymology*; D014640:Vancomycin / Q000096:biosynthesis*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D011994:Recombinant Proteins; C070700:VanH protein, Enterococcus faecium; D014640:Vancomycin; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase; D010072:Oxamic Acid