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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12440191/
• 关键词: cysteine-sulfinic acid; cysteine-sulfenic acid; non-heme protein; post-translational modification; nitrile hydratase; thiocyanate hydrolase; metallochapeorne; non-corrin cobalt protein; 非ヘム鉄蛋白質; 非ヘム鉄タンパク質

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.

硝酸盐水合物（NHASE）是含有非血红素或非corn钴的金属酶，并将riles的水合催化为相关的酰胺。氧化的半胱氨酸配体，半胱氨酸 - 硫酸（CYS-SOH）和半胱氨酸 - 硫酸含量（CYS-SO2H），我们研究了新硝酸盐水解酶家族中新型金属结合基序的功能。 - 据报道，我们发现的Ki值是正常的，并以2-苯甲酸2-丙基2-抗氧化物的杂质抑制，质子化（IBN）被报告为竞争性抑制剂。 。结果表明，αcys114的cys-soh soh结构对于催化活性至关重要已经将tase扔到了co型Nhase的一个疗程中，我们在e.coli中构建了Apo-Scnase系统，并进行了一些结晶。

项目成果

Endo,Isao: "What evidences were elucidated about photoreactive nitrile hydratase?"Journal Molecular Catalysis B : Enzymatic. 10. 81-86 (2000)

Endo,Isao：“关于光反应性腈水合酶阐明了哪些证据？”《分子催化 B 杂志：酶学》。

Endo, Isao: "What evidences were elucidated about photo-reactive nitrile hydratase?"Journal Molecular Catalysis B : Enzymatic. Vol.10. 81-86 (2000)

Endo, Isao：“关于光反应性腈水合酶阐明了哪些证据？”《分子催化 B 杂志：酶学》。

Tsujimura, Masanari: "A novel inhibitor for Fe-type nitrile hydratase 2-cyano-2-propyl hydroperoxide"Journal of American Chemical Society. 125. 11532-11538 (2003)

Tsujimura，Masanari：“Fe型腈水合酶2-氰基-2-丙基氢过氧化物的新型抑制剂”美国化学会杂志。

Piersma, Sander R.: "Arginine 56 mutation in beta subunit of nitrile hydratase : importance of hydrogen bonding to the non-heme iron center"Journal of Bioinorganic Chemistry. Vol.80. 283-288 (2000)

Piersma, Sander R.：“腈水合酶 β 亚基中的精氨酸 56 突变：氢键对非血红素铁中心的重要性”生物无机化学杂志。

Endo, Isao: "Fe-type nitrile hydratase"Journal of Bioinorganic chemistry. Vol.83. 247-253 (2001)

Endo，Isao：“Fe型腈水合酶”生物无机化学杂志。