Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase
Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase
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DOI：
10.1016/j.jbiosc.2013.01.013
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发表时间：
2013-07-01
影响因子：
2.8
通讯作者：
Odaka, Masafumi
中科院分区：
文献类型：
--
作者：
Yamanaka, Yasuaki;Arakawa, Takatoshi;Odaka, Masafumi
Thiobacillus thioparus THI115的硫氰酸酯水解酶（SCNase）是一种含钴（Co）的酶，可以催化硫氰酸酯（SCN-）水解为羰基硫醚和氨，硫氰酸酯是焦炉工厂废水的主要成分。尽管SCNase与co型腈水合酶（NHase）具有高度的结构相似性，包括独特的Co3+催化中心和两个氧化的Cys配体，但SCNase和NHase都只催化它们自己的底物。基于这些酶的底物结合袋的差异，在co型nase中，侧链向袋方向延伸的SCNase的β Arg90和γ Arg136分别被相应的氨基酸残基Phe和Trp取代。SCNase β Arg90和SCNase γ Arg136突变体均无SCN-水解活性，但能催化腈的水化作用。估计的k（cat）值（类似于2s(-1)）相当于co型nase用于腈水化的约0.2%，相当于野生型SCNase用于SCN-水解的约3%。SCNase γ R136W的晶体结构与野生型基本相同，包括Co3+中心有Cys氧化；由于突变残基侧链的构象改变，底物袋的大小增大。对野生型和突变型SCNase以及co型nase的底物结合袋周围环境差异的讨论强烈表明，位于Co3+中心顶部的β Arg90和γ Arg136主要控制SCNase的底物选择性。(C) 2013，生物技术学会，日本。版权所有。
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, beta Arg90 and gamma Arg136 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 beta Arg90 and SCNase gamma Arg136 mutants showed no SCN- hydrolysis activity but did catalyze the hydration of nitriles. The estimated k(cat) values (similar to 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 gamma R136W 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 beta Arg90 and gamma Arg136, positioned at the top of the Co3+ center, predominantly control the substrate selectivity of SCNase. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.