School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
Biochem J. 2013 Apr 15;451(2):217-26. doi: 10.1042/BJ20121715.
The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser(1β), is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.
已确定来自假单胞菌 N176 的戊二酰-7-ACA（7-氨基头孢烷酸）酰化酶的野生型和蛋白质的双突变体（H57βS/H70βS）的晶体结构，该突变体在头孢菌素 C 上相对于戊二酰-7-氨基头孢烷酸显示出增强的催化效率。这些结构显示由一个α-链（229 个残基）和一个β-链（543 个残基）组成的杂二聚体，具有一个深腔，构成了活性部位。野生型和突变型结构的比较提供了对观察到的头孢菌素 C 相对于戊二酰-7-氨基头孢烷酸的增强特异性的分子原因的深入了解，并为进一步进化出更好的酶变体提供了基础。亲核催化丝氨酸残基 Ser（1β）位于活性部位腔的底部。电子密度显示出与催化丝氨酸残基共价结合的配体，从而形成了四面体加合物。这被认为模拟了酶的过渡态，无论是成熟步骤还是底物的催化。酶的过渡态构象的视图提供了对底物结合和催化机制的重要见解。
