Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase

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• 作者：Yasuaki Yamanaka¹ ; Takatoshi Arakawa¹ ; Toshinori Watanabe¹ ; Satoshi Namima¹ ; Masa Sato¹ ; Shota Hori¹ ; Akashi Ohtaki¹ ; Keiichi Noguchi² ; Yoko Katayama³ ; Masafumi Yohda¹ ; Masafumi Odaka¹ ; ^{modaka@cc.tuat.ac.jp} ; ^{masafumi.odaka@yohda.net}
• 关键词：Thiocyanate hydrolase ; Nitrile hydratase ; Non-corrin cobalt ; Substrate selectivity ; Hydrolysis
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2013
• 出版时间：July, 2013
• 年：2013
• 卷：116
• 期：1
• 页码：22-27
• 全文大小：1071 K

文摘

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt (Co)-containing enzyme that catalyzes the hydrolysis of thiocyanate (SCN^?), a major component of wastewater from coke oven factories, to carbonyl sulfide and ammonia. Although SCNase exhibits high structural similarities to Co-type nitrile hydratase (NHase), including a unique Co³⁺ catalytic center with two oxidized Cys ligands, both SCNase and NHase exclusively catalyze only their own substrates. Based on the differences in the substrate-binding pockets of these enzymes, ¦ÂArg90 and ¦ÃArg136 of SCNase, with side chains extending toward the pocket, were separately substituted with Phe and Trp, the corresponding residues, respectively, in Co-type NHase. Both SCNase ¦ÂArg90 and SCNase ¦ÃArg136 mutants showed no SCN^? hydrolysis activity but did catalyze the hydration of nitriles. The estimated k_cat values (¡«2?s^?1) corresponded to approximately 0.2 % of that of Co-type NHase for nitrile hydration and approximately 3 % of that of wild-type SCNase for SCN^? hydrolysis. The crystal structure of SCNase ¦ÃR136W is essentially identical to that of the wild-type, including the Co³⁺ center having Cys oxidations; the size of the substrate pocket was enlarged because of conformational changes on the side chains of the mutated residue. Discussion of the difference in the environments around the substrate-binding pockets among the wild-type and mutant SCNases and Co-type NHase strongly suggests that ¦ÂArg90 and ¦ÃArg136, positioned at the top of the Co³⁺ center, predominantly control the substrate selectivity of SCNase.