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.