Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity

TitleGlutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity
Publication TypeJournal Article
Year of Publication2005
AuthorsNemeria, N, Tittmann K, Joseph E, Zhou L, Vazquez-Coll MB, Arjunan P, Hubner G, Furey W, Jordan F
JournalJournal of Biological Chemistry
Volume280
Pagination21473-21482
Date PublishedJun
Type of ArticleArticle
ISBN Number0021-9258
Accession Numberhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000229438800068
KeywordsACETOHYDROXYACID SYNTHASE, ACID-BASE, ANGSTROM, BENZOYLFORMATE DECARBOXYLASE, COMPLEX E1 SUBUNIT, DIPHOSPHATE, ENZYMES, MULTIENZYME COMPLEX, REDUCTIVE ACETYLATION, RESOLUTION, SUBSTRATE
Abstract

The residue Glu(636) is located near the thiamine diphosphate ( ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K-d,K-ThDP = 4.34 mu M and K-m,K- ThDP = 11 mu M were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1', 4'- iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)- acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp(28)-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity ( re or si) vis a vis pyruvate. The tryptic peptide map ( mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.

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Alternate JournalJ. Biol. Chem.