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Titolo:
D-2-HYDROXY-4-METHYLVALERATE DEHYDROGENASE FROM LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS .2. MUTAGENIC ANALYSIS OF CATALYTICALLY IMPORTANT RESIDUES
Autore:
BERNARD N; JOHNSEN K; GELPI JL; ALVAREZ JA; FERAIN T; GARMYN D; HOLS P; CORTES A; CLARKE AR; HOLBROOK JJ; DELCOUR J;
Indirizzi:
UNIV CATHOLIQUE LOUVAIN,DEPT BIOL,GENET UNIT,PL CROIX SUD 5,6 B-1348 LOUVAIN BELGIUM UNIV CATHOLIQUE LOUVAIN,DEPT BIOL,GENET UNIT B-1348 LOUVAIN BELGIUM UNIV BRISTOL,DEPT BIOCHEM BRISTOL AVON ENGLAND UNIV BRISTOL,MOL RECOGNIT CTR BRISTOL AVON ENGLAND UNIV BARCELONA,DEPT BIOQUIM & BIOL MOL BARCELONA SPAIN
Titolo Testata:
European journal of biochemistry
fascicolo: 1, volume: 244, anno: 1997,
pagine: 213 - 219
SICI:
0014-2956(1997)244:1<213:DDFL>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
D-LACTATE DEHYDROGENASE; LIGAND-BINDING SITES; D-2-HYDROXYISOCAPROATE DEHYDROGENASE; FORMATE DEHYDROGENASE; ESCHERICHIA-COLI; PLANTARUM; CLONING; GENE; RESOLUTION; FRAMEWORK;
Keywords:
D-2-HYDROXY-4-METHYLVALERATE DEHYDROGENASE; LACTOBACILLUS DELBRUECKII BULGARICUS; SUBSTRATE INHIBITION; D-LACTATE DEHYDROGENASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
23
Recensione:
Indirizzi per estratti:
Citazione:
N. Bernard et al., "D-2-HYDROXY-4-METHYLVALERATE DEHYDROGENASE FROM LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS .2. MUTAGENIC ANALYSIS OF CATALYTICALLY IMPORTANT RESIDUES", European journal of biochemistry, 244(1), 1997, pp. 213-219

Abstract

Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that asingle histidine residue was involved in catalysis. Since H296 is theonly conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold compared with the wild-type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar andlarge reductions in their k(cat)/K-m ratios (200-800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the alpha-carboxyl group of the substrate and it was changed to lysine. The R235Kmutant showed a 104-fold reduced k(cat)/K-m due to both an increased K-m and a reduced k(cat) for 2-oxo-4-methylvalerate. Thus R235 plays arole in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases. Finally, we constructed the H205Q mutant to testthe role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7-fold increased k(cat) and a doubled catalytic efficiency at pH 5, was as sensitive todiethylpyrocarbonate as the wildtype but showed a sevenfold increasedK-m for NADH and a 100-fold increase in K-d for NADH together with 10-30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5.

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Documento generato il 03/12/20 alle ore 14:32:37