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

具有氧化半胱氨酸残基的新型金属结合基序的功能

基本信息

批准号：
12440191
负责人：
ODAKA Masafumi
金额：
$ 4.16万
依托单位：
RIKEN
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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.

氰基水合酶(NHase)是一类含有非血红素或非科林钴活性中心的金属酶，催化各种氰化物水合生成相应的酰胺类化合物。我们已经证明了红球菌的Fe型氨基转移酶。N771有一个新的金属结合基序，含有两个氧化半胱氨酸配体，半胱氨酸-磺酸(Cys-SOH)和半胱氨酸-亚磺酸(Cys-SO2H)。在本研究中，我们研究了氰基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的功能被报道为竞争性抑制物，其Ki值为5μM。我们发现，正品异丁腈正常水合，商品异丁腈中的杂质2-氰基-2-丙基过氧化氢(CPx)对氨酶活性有强烈的抑制作用。CPX特异地氧化金属结合基序中的Cys-SOH配体，以不可逆失活该酶。丁酸(BA)是NHase的稳定剂，被用于纯化、储存和大多数实验报道。我们研究了BA对NHase的稳定作用。结果表明，BA对αCys114-SOH的有氧氧化为Cys-SO2H有保护作用。这两个结果都证明了αCys114的半胱氨酸结构对催化活性是必不可少的。2.我们发现，半胱氨酸修饰不仅在Co型氨酶中保守，而且在硫氰酸盐水解酶中也是保守的，其氨基酸序列与氨酶非常保守。我们还证明了SCNase是Co型NHase的一个新成员。我们构建了apo-SCNase在大肠杆菌中的表达系统，并对其进行了结晶。脱氧核糖核酸酶的X射线晶体结构分析目前正在进行中。