myo-inositol oxygenase:: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and D-chiro-inositol

被引:71
作者
Arner, RJ
Prabhu, KS
Thompson, JT
Hildenbrandt, GR
Liken, AD
Reddy, CC
机构
[1] Penn State Univ, Dept Vet Sci, University Pk, PA 16802 USA
[2] Penn State Univ, Ctr Mol Toxicol & Carcinogenesis, University Pk, PA 16802 USA
关键词
diabetes; D-glucuronate; kidney; mono-oxygenase; pig;
D O I
10.1042/bj3600313
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Fe2+ 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.
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页码:313 / 320
页数:8
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