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

腈水合酶（Nitrile hydratases，简称NHase）是一类含有非血红素或非咕啉钴活性中心的金属酶，催化各种腈水合为相应的酰胺。我们发现红球菌N771的Fe型腈水合酶具有一个新的金属结合基序，该基序包含两个氧化的半胱氨酸配体，半胱氨酸-亚磺酸（Cys-SOH）和半胱氨酸-亚磺酸（Cys-SO2 H）。本文研究了腈水合酶家族中新的金属结合基序的功能：1.氧化半胱氨酸配体-异丁腈（IBN）的功能已被报道为竞争性抑制剂，Ki值为5 μM。我们发现，真实的IBN是正常水合的，并且市售IBN中存在的杂质2-氰基-2-丙基氢过氧化物（Cpx）强烈抑制了腈水合酶活性。Cpx特异性地氧化金属结合基序中的Cys-SOH配体，使酶不可逆地稳定。已知正丁酸（BA）是腈水合酶的稳定剂，并用于纯化、储存和大多数实验报道。我们研究了BA如何稳定NHase。结果表明，BA能保护α Cys 114-SOH不被好氧氧化为Cys-SO_2 H。结果表明，α Cys 114的Cys-SOH结构对酶的催化活性至关重要。2.发现Cys-SO2 H结构不仅在Co型氨合酶中是保守的，而且在氨基酸序列与氨合酶高度保守的硫氰酸盐水解酶（SCNase）中也是保守的。我们还表明，SCNase是一个新的成员的Co型腈水合酶。构建了apo-SCNase在大肠杆菌中的表达系统。coli，并成功地进行了结晶。apo-SCNase的X射线晶体结构分析目前正在进行中。

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.