Function of a novel metal-binding motif having oxidized cysteine residues

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Nitrile hydratases (NHase) are metalloenzymes containing a non-heme or a non-corrin cobalt active center and catalyze the hydration of various nitriles to the corresponding amides. We have shown that Fe-type NHase of Rhodococcus sp. N771 has a novel metal-binding motif containing two oxidized cysteine ligands, cysteine-sulfenic acid (Cys-SOH) and cysteine-sulfinic acid (Cys-SO2H). In the present research, we studied functions of the novel metal-binding motif in nitrile hydratase family.1.Function of the oxidized cysteine ligands-Isobutyronitrile (IBN) had been reported as a competitive inhibitor with a Ki value of 5 μM. We found that authentic IBN was hydrated normally and that the impurity present in commercially available IBN, 2-cyano-2-propyl hydroperoxide (Cpx) inhibited NHase activity strongly. Cpx specifically oxidized the Cys-SOH ligand in the metal-binding motif, to inactivate the enzyme irreversibly n-Butyric acid (BA) is known to a stabilizing agent of NHase and used for purification, storage and most experiments reported. We studied how BA stabilizes NHase. We showed that BA protected αCys114-SOH from aerobic oxidation to Cys-SO_2H. Both results results demonstrated that the Cys-SOH structure of αCys114 is essential for the catalytic activity.2.We revealed that the Cys-SO2H modification was conserved not only in Co-type NHase but also in thiocyanate hydrolase (SCNase) whose amino acid sequences were well conserved with NHases. We also have shown that SCNase is a novel member of Co-type NHase. We constructed the expression system of apo-SCNase in E..coli, and succeeded in its crystallization. X-ray crystal structure analysis of apo-SCNase is currently underway.