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Titolo:
Molecular mechanism of VanHst, an alpha-ketoacid dehydrogenase required for glycopeptide antibiotic resistance from a glycopeptide producing organism
Autore:
Marshall, CG; Zolli, M; Wright, GD;
Indirizzi:
McMaster Univ, Dept Biochem, Antimicrobial Res Ctr, Hamilton, ON L8N 3Z5, Canada McMaster Univ Hamilton ON Canada L8N 3Z5 tr, Hamilton, ON L8N 3Z5, Canada
Titolo Testata:
BIOCHEMISTRY
fascicolo: 26, volume: 38, anno: 1999,
pagine: 8485 - 8491
SICI:
0006-2960(19990629)38:26<8485:MMOVAA>2.0.ZU;2-6
Fonte:
ISI
Lingua:
ENG
Soggetto:
D-LACTATE DEHYDROGENASE; VANCOMYCIN-RESISTANCE; STREPTOMYCES-TOYOCAENSIS; SUBSTRATE-BINDING; VANA; CATALYSIS; LIGASES; GENES;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
17
Recensione:
Indirizzi per estratti:
Indirizzo: Wright, GD McMaster Univ, Dept Biochem, Antimicrobial Res Ctr, 1200 Main St W, Hamilton, ON L8N 3Z5, Canada McMaster Univ 1200 Main St W Hamilton ON Canada L8N 3Z5 Canada
Citazione:
C.G. Marshall et al., "Molecular mechanism of VanHst, an alpha-ketoacid dehydrogenase required for glycopeptide antibiotic resistance from a glycopeptide producing organism", BIOCHEM, 38(26), 1999, pp. 8485-8491

Abstract

The vancomycin resistance enzyme VanH is an a-ketoacid dehydrogenase that stereospecifically reduces pyruvate to D-lactate, which is required for thesynthesis 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 pyruvatereduction with k(cat)/K-m = 1.3 x 10(5) M-1 s(-1) and has a more restricted cr-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 pK(a) of the catalytic His in other D-lactate dehydrogenases, was found to fulfill a similar role in VanHst, lowering a pK(a) value of k(cat)/K-m nearly 2 units. These results now provide the framework for additional structureand inhibitor design work on the VanH family of antibiotic resistance enzymes.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 03/12/20 alle ore 15:55:09