Wong Brian J, Gerlt John A
Departments of Biochemistry and Chemistry, University of Illinois, Urbana, Illinois 61801, USA.
Biochemistry. 2004 Apr 27;43(16):4646-54. doi: 10.1021/bi0360307.
Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole. In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned. Focusing on the orthologues from Pseudomonas aeruginosa and P. putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA. The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase. Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues. Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.
烯酰辅酶A水合酶（巴豆酸酶）超家族的成员催化不同的整体反应，这些反应采用由共享结构支架提供的共同催化策略；底物通常是辅酶A的酰基酯，中间体通常是由保守的氧负离子洞稳定的硫酯烯醇化物阴离子。在许多细菌基因组中，含有大鼠线粒体巴豆酸酶中酸/碱催化剂Glu164的同源物但不含有Glu144同源物的直系同源成员由功能尚未确定的操纵子编码。以铜绿假单胞菌和恶臭假单胞菌的直系同源物为研究对象，我们确定这些操纵子编码亮氨酸分解代谢中的酶，其中未知酶被指定为（3S）-甲基谷氨酰辅酶A水合酶（MGCH），它催化（E）-3-甲基谷氨酰辅酶A向（3S）-羟甲基戊二酰辅酶A的顺式水合反应。细菌MGCH利用单个活性位点残基催化烯酰辅酶A水合反应的发现，与典型的巴豆酸酶以及最近鉴定的在巴豆酸酶中使用Glu144和Glu164同源物的哺乳动物MGCH形成对比。缺乏γ-羧酸盐的底物类似物已被证明是该酶的竞争性抑制剂，并且用谷氨酸替代Glu144的“缺失”同源物并不能引入对底物类似物的水合酶活性。因此，细菌MGCH可能提供了一个机会主义进化的例子，其中底物的羧酸盐基团在由巴豆酸酶和真核MGCH催化的反应中在功能上取代了活性位点谷氨酸残基之一。
