Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics(2012)

引用 19|浏览22
暂无评分
摘要
The joint substitution of three active-site residues in Escherichia coli l-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 105-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, kcat′ for desulfination of l-cysteine sulfinate increased to 0.5s−1 (from 0.05s−1 in wild-type enzyme), whereas kcat′ for transamination of the same substrate was reduced from 510s−1 to 0.05s−1. Similarly, kcat′ for β-decarboxylation of l-aspartate increased from<0.0001s−1 to 0.07s−1, whereas kcat′ for transamination was reduced from 530s−1 to 0.13s−1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme–substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme–substrate intermediates by bulk water.
更多
查看译文
关键词
EcAspAT,PLP,PMP
AI 理解论文
溯源树
样例
生成溯源树,研究论文发展脉络
Chat Paper
正在生成论文摘要