Myo-inositol oxygenase (MIOX) catalyzes the first step in the only known pathway in humans for breakdown of myo-inositol (Ml), the sugar backbone of cell-signaling phosphoinositides. Evidence suggests that increased expression or activity of MIOX may contribute to pathologies commonly associated with diabetes mellitus, marking MIOX as a potential drug target. Our preliminary data show that the MIOX reaction proceeds via an unprecedented chemical mechanism. A non-heme diiron cluster in its mixed-valent, ll/lll, oxidation state, which probably coordinates one or more oxygen atoms of the substrate, reacts with molecular oxygen to generate a formally diiron(lll/lll)-superoxide complex, which abstracts hydrogen from the substrate. We seek to determine by biophysical and biochemical methods: (1) the structure of this unique enzyme and its diiron cofactor; (2) how the protein promotes initial formation and stability of the active, mixed-valent form (which is unstable in many other non-heme diiron proteins); (3) how the substrate interacts with the protein and cofactor; (4) whether and how this interaction activates the cofactor or substrate (or both) for subsequent reaction with O2; and (5) the nature and rate of each chemical step leading from addition of oxygen to the enzyme*substrate complex through release of the product along with the structures of two reactive intermediates that we have already discovered and additional intermediates that remain to be discovered in this sequence. By so doing, we hope to provide the necessary tools for rational design of MIOX inhibitors that could be useful in combating complications from diabetes mellitus. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK074641-02
Application #
7340497
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Sechi, Salvatore
Project Start
2007-01-15
Project End
2010-12-31
Budget Start
2008-01-01
Budget End
2008-12-31
Support Year
2
Fiscal Year
2008
Total Cost
$280,115
Indirect Cost
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
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Snyder, Rae Ana; Bell 3rd, Caleb B; Diao, Yinghui et al. (2013) Circular dichroism, magnetic circular dichroism, and variable temperature variable field magnetic circular dichroism studies of biferrous and mixed-valent myo-inositol oxygenase: insights into substrate activation of O2 reactivity. J Am Chem Soc 135:15851-63
Dassama, Laura M K; Yosca, Timothy H; Conner, Denise A et al. (2012) O(2)-evolving chlorite dismutase as a tool for studying O(2)-utilizing enzymes. Biochemistry 51:1607-16
van der Donk, Wilfred A; Krebs, Carsten; Bollinger Jr, J Martin (2010) Substrate activation by iron superoxo intermediates. Curr Opin Struct Biol 20:673-83
Bollinger Jr, J Martin; Diao, Yinghui; Matthews, Megan L et al. (2009) myo-Inositol oxygenase: a radical new pathway for O(2) and C-H activation at a nonheme diiron cluster. Dalton Trans :905-14
Bollinger Jr, J Martin; Krebs, Carsten (2007) Enzymatic C-H activation by metal-superoxo intermediates. Curr Opin Chem Biol 11:151-8