title: Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase.
abstract: 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, βArg90 and γArg136 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 βArg90 and SCNase γArg136 mutants showed no SCN⁻ hydrolysis activity but did catalyze the hydration of nitriles. The estimated kcat 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 γR136W 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 βArg90 and γArg136, positioned at the top of the Co³⁺ center, predominantly control the substrate selectivity of SCNase.
authors: Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
authors: Yasuaki
authors: Y
authors: Yamanaka
authors: Takatoshi
authors: T
authors: Arakawa
authors: Toshinori
authors: T
authors: Watanabe
authors: Satoshi
authors: S
authors: Namima
authors: Masa
authors: M
authors: Sato
authors: Shota
authors: S
authors: Hori
authors: Akashi
authors: A
authors: Ohtaki
authors: Keiichi
authors: K
authors: Noguchi
authors: Yoko
authors: Y
authors: Katayama
authors: Masafumi
authors: M
authors: Yohda
authors: Masafumi
authors: M
authors: Odaka
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry*; D055162:Biocatalysis; D003035:Cobalt / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013855:Thiobacillus / Q000201:enzymology
chemical_list: D003035:Cobalt; D001120:Arginine; D006867:Hydrolases; C074369:thiocyanate hydrolase; D006836:Hydro-Lyases; C048831:nitrile hydratase; D003545:Cysteine