Institute of Biochemistry, University of Leipzig, Leipzig, Germany.
FEBS Lett. 2011 May 6;585(9):1317-21. doi: 10.1016/j.febslet.2011.03.063. Epub 2011 Apr 6.
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.
戊二酰辅酶 A（CoA）脱氢酶（GDH）是酰基辅酶 A 脱氢酶，通常将底物脱氢和脱羧为巴豆酰辅酶 A。在一些厌氧菌中，存在非脱羧 GDH，其不脱羧而释放戊二酰辅酶 A（2,3-脱水戊二酰辅酶 A）。通过对产电菌 Geobacter metallireducens 中形成巴豆酰辅酶 A 的 GDH 基因的定点突变，研究了脱羧和非脱羧 GDH 的不同机制。交换参与底物羧酸盐结合的单个氨基酸会损害脱羧步骤，导致相对戊二酰辅酶 A：巴豆酰辅酶 A 形成率为 1：1（S97A）或 13：1（Y370A）。Y370V+S97A 双突变体中形成的戊二酰辅酶 A 总量最大。所得结果表明，不变的去质子化 Tyr 对于优化脱羧 GDH 中 CO2 的离去基团势能起着至关重要的作用。
