Conversion of a decarboxylating to a non-decarboxylating glutaryl-coenzyme A dehydrogenase by site-directed mutagenesis.

Jörg Schaarschmidt, Simon Wischgoll, Hans-Jörg Hofmann, Matthias Boll, Jörg Schaarschmidt, Simon Wischgoll, Hans-Jörg Hofmann, Matthias Boll

Index: FEBS Lett. 585 , 1317-1321, (2011)

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Abstract

Glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are acyl-CoA dehydrogenases, which usually dehydrogenate and decarboxylate the substrate to crotonyl-CoA. In some anaerobic bacteria, non-decarboxylating GDHs exist that release glutaconyl-CoA (2,3-dehydroglutaryl-CoA) without decarboxylation. The differing mechanisms of decarboxylating and non-decarboxylating GDHs were investigated by site-directed mutagenesis of the gene coding for the crotonyl-CoA-forming GDH from Geobacter metallireducens. Exchange of single amino acids involved in substrate carboxylate binding impaired the decarboxylation step, resulting in relative glutaconyl-CoA:crotonyl-CoA formation rates of 1:1 (S97A) or 13:1 (Y370A). The total amount of glutaconyl-CoA formed was maximal in the Y370V+S97A double mutant. The results obtained indicate that an invariant deprotonated Tyr plays a crucial role for optimizing the leaving group potential of CO(2) in decarboxylating GDHs.Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.