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Allen Orville - NAD(P)H Oxidase (NAD(P)H-Ox)

Allen Orville 's research interests explained in more detail:

Lactobacillus sanfranciscensis alleviates oxidative stress and regenerates NAD(P)+ for glycolysis via the FAD-dependent NAD(P)H oxidase. However, the enzyme does not release hydrogen peroxide during steady state turnover, but rather forms two water molecules from two equivalents of either NADH or NADPH. Our 1.8 Å resolution structure reveals Cys42 adjacent to the FAD and oxidized to at least the sulfenic acid state (R-SOH). Thus the enzyme uses a redox-active cysteine residue that alternates between the thiol/thiolate and the sulfenic acid states to achieve catalysis. Our structural analysis also revealed the presence of an endogenous 'mystery' ligand that copurified and crystallized with the resting enzyme. It binds in the NAD(P)H domain and partially occludes the FAD re-face. Therefore, its presence also likely influences the binding of substrate NAD(P)H molecules. The retention times from reverse-phase HPLC and the absorption at 260 and/or 450 nm for the peaks associated with the ligands released from denatured enzyme are consistent with FAD, but the second major peak does not have retention times similar to NAD+, NADH, NADP+ or NADPH. Results from MALDI-TOF mass spectral analysis of the released ligands identify parent ion species with masses consistent with FAD and ADP. Therefore, we conclude ADP is the tightly bound ligand and hypothesize that it remains bound during turnover and thus provides a structural basis for the lack of substrate specificity exhibited by the enzyme.

  • Supported by: American Chemical Society, Petroleum Research Fund (40310-G4), A.M.O. (PI) and
    American Heart Association Grant in Aid (0555286B), A.M.O. (PI).
  • Lountos G.T., Riebel B.R., Wellborn W.B., Bommarius A.S. and Orville A.M.
    Crystallization and preliminary analysis of a water-forming NADH oxidase from Lactobacillus sanfranciscensis.
    Acta Crystallography, D60:2044–2047 (2004).   PubMed
  • Lountos G.T., Jiang R., Wellborn W.B., Thaler T.L., Bommarius A.S. and Orville A.M.
    The Crystal Structure of NAD(P)H Oxidase from Lactobacillus sanfranciscensis: Insights into the Conversion of O2 into Two Water Molecules by the Flavoenzyme.
    Biochemistry, 45:9648-9659 (2006).   PubMed
    PDB file: 2CDU