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Titolo:
Binding and activation of thiamin diphosphate in acetohydroxyacid synthase
Autore:
Bar-Ilan, A; Balan, V; Tittman, K; Golbik, R; Vyazmensky, M; Hubner, G; Barak, Z; Chipman, DM;
Indirizzi:
Ben Gurion Univ Negev, Dept Life Sci, IL-84105 Beer Sheva, Israel Ben Gurion Univ Negev Beer Sheva Israel IL-84105 4105 Beer Sheva, Israel Univ Halle Wittenberg, Dept Biochem, D-06099 Halle Saale, Germany Univ Halle Wittenberg Halle Saale Germany D-06099 9 Halle Saale, Germany
Titolo Testata:
BIOCHEMISTRY
fascicolo: 39, volume: 40, anno: 2001,
pagine: 11946 - 11954
SICI:
0006-2960(20011002)40:39<11946:BAAOTD>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
SITE-DIRECTED MUTAGENESIS; MOBILIS PYRUVATE DECARBOXYLASE; ESCHERICHIA-COLI; ACID SYNTHASE; ZYMOMONAS-MOBILIS; CRYSTAL-STRUCTURE; ANGSTROM RESOLUTION; DEPENDENT ENZYME; ISOZYME-III; LACTOBACILLUS-PLANTARUM;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
44
Recensione:
Indirizzi per estratti:
Indirizzo: Chipman, DM Ben Gurion Univ Negev, Dept Life Sci, POB 653, IL-84105 Beer Sheva, Israel Ben Gurion Univ Negev POB 653 Beer Sheva Israel IL-84105 rael
Citazione:
A. Bar-Ilan et al., "Binding and activation of thiamin diphosphate in acetohydroxyacid synthase", BIOCHEM, 40(39), 2001, pp. 11946-11954

Abstract

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation or pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactorfor proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitudeand decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg2+ in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg2+. While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg2+, D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K+. These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP, changesin the ligands to ThDP can have interesting and unexpected effects on the reaction.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 02/04/20 alle ore 09:43:08