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Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

ABSTRACT: myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI complex reacts rapidly and reversibly with O2 to form an intermediate, G, with a g = (2.05, 1.98, 1.90) EPR signal. The rhombic g-tensor and observed hyperfine coupling to 57Fe are rationalized in terms of a (superoxo)diiron(III/III) structure with coordination of the superoxide to a single iron. G decays to H, the intermediate previously detected in the reaction with unlabeled substrate. This step is associated with a kinetic isotope effect of > or =5, showing that the superoxide-level complex does indeed cleave a C-H(D) bond of MI.

SUBMITTER: Xing G

PROVIDER: S-EPMC1458843 | biostudies-other | 2006 Apr

REPOSITORIES: biostudies-other

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Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

Xing Gang G Diao Yinghui Y Hoffart Lee M LM Barr Eric W EW Prabhu K Sandeep KS Arner Ryan J RJ Reddy C Channa CC Krebs Carsten C Bollinger J Martin JM

Proceedings of the National Academy of Sciences of the United States of America 20060410 16

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI ...[more]

PMID: 16606846

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Project description:Renal-specific oxidoreductase/myo-inositol oxygenase (RSOR/MIOX) catabolizes myo-inositol and is implicated in the pathogenesis of diabetic nephropathy. How high glucose (HG) ambience up-regulates its expression and enzyme activity was investigated. MIOX up-regulation was associated with an increase in enzyme activity, which was reduced to basal levels with phosphatase treatment. Using phosphothreonine, protein kinase A (PKA), and PKC substrate antibodies, analyses of kidney lysates of diabetic animals and LLC-PK1/HK-2 cells subjected to HG ambience indicated MIOX to be a phosphoprotein. Kinase phosphorylated recombinant RSOR/MIOX proteins had increased activity confined to exons 2-5. Mutants with substituted phosphorylation sites had a minimal increase in activity. Treatment of cells with PKC, PKA, and PDK1 kinase activators increased activity, whereas inhibitors reduced it. Inhibitors also reduced the phosphorylation and activity of MIOX induced by HG. Besides HG, exposure of cells to oxidants H(2)O(2) and methylglyoxal up-regulated MIOX expression and its phosphorylation and activity, whereas antioxidants N-acetylcysteine, β-naphthoflavone, and tertiary butyl hydroquinone reduced MIOX expression. Treatment with HG or oxidants or overexpression of MIOX induced nuclear translocation of redox-sensitive transcription factor Nrf2, which binds to antioxidant response elements of various promoters. Promoter analyses revealed an increase in luciferase activity with HG and oxidants. Analyses of antioxidant response elements and carbohydrate response elements revealed an accentuation of DNA-protein interactions with oxidants and under HG ambience. ChIP-PCR and immunofluorescence studies revealed nuclear translocation of carbohydrate response element-binding protein. These findings suggest that phosphorylation of RSOR/MIOX enhances its activity, which is augmented by HG via transcriptional/translational events that are also modulated by diabetes-related pathobiological stresses.

Dataset Information

Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

Publications

Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

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