myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and D-chiro-inositol.
myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and D-chiro-inositol.
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DOI：
10.1042/0264-6021:3600313
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发表时间：
2001-12
期刊：
影响因子：
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通讯作者：
R. Arner;K. S. Prabhu;Jerry T. Thompson;G. Hildenbrandt;A. Liken;C. Reddy
中科院分区：
文献类型：
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作者：
R. Arner;K. S. Prabhu;Jerry T. Thompson;G. Hildenbrandt;A. Liken;C. Reddy
肌醇加氧酶 (MIOX) 催化肌醇分解代谢的唯一途径中的第一个关键步骤，该分解代谢主要发生在肾脏中。该酶是一种非血红素铁酶，可催化肌醇的环裂解并掺入单个氧原子。从猪肾文库中分离得到全长cDNA，开放阅读框为849 bp，相应的蛋白质亚基分子量为32.7 kDa。 cDNA 在细菌 pET 表达系统中表达，并从细菌裂解物中纯化出活性重组 MIOX，使其电泳均质。纯化的酶表现出与天然酶相同的催化特性，K(m)和k(cat)值分别为5.9 mM和11 min(-1)。 pI 估计为 4.5。用 1 mM Fe(2+) 和 2 mM 半胱氨酸预孵育对于酶的活性至关重要。 D-手性肌醇是一种肌醇异构体，是重组 MIOX 的底物，估计 K(m) 为 33.5 mM。肌醇和D-手性肌醇都与糖尿病的发病机制有关。因此，对 MIOX 表达调节的理解清楚地代表了糖尿病病因学以及各种细胞内磷酸肌醇和关键信号通路的控制的潜在窗口。
myo-Inositol oxygenase (MIOX) catalyses the first committed step in the only pathway of myo-inositol catabolism, which occurs predominantly in the kidney. The enzyme is a non-haem-iron enzyme that catalyses the ring cleavage of myo-inositol with the incorporation of a single atom of oxygen. A full-length cDNA was isolated from a pig kidney library with an open reading frame of 849 bp and a corresponding protein subunit molecular mass of 32.7 kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with K(m) and k(cat) values of 5.9 mM and 11 min(-1) respectively. The pI was estimated to be 4.5. Preincubation with 1 mM Fe(2+) and 2 mM cysteine was essential for the enzyme's activity. D-chiro-Inositol, a myo-inositol isomer, is a substrate for the recombinant MIOX with an estimated K(m) of 33.5 mM. Both myo-inositol and D-chiro-inositol have been implicated in the pathogenesis of diabetes. Thus an understanding of the regulation of MIOX expression clearly represents a potential window on the aetiology of diabetes as well as on the control of various intracellular phosphoinositides and key signalling pathways.