title: Conversion of a decarboxylating to a non-decarboxylating glutaryl-coenzyme A dehydrogenase by site-directed mutagenesis.
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.
authors.affiliation: Institute of Biochemistry, University of Leipzig, Leipzig, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Schaarschmidt
authors.forename: Simon
authors.initials: S
authors.lastname: Wischgoll
authors.forename: Hans-Jörg
authors.initials: HJ
authors.lastname: Hofmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Boll
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D003653:Decarboxylation; D045302:Geobacter / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D050770:Glutaryl-CoA Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; C015901:glutaryl-coenzyme A; D014443:Tyrosine; C032423:glutaconyl-coenzyme A; C010701:crotonyl-coenzyme A; D050770:Glutaryl-CoA Dehydrogenase