The long-term objective of this application is to elucidate the roles of a novel group of proteins related in sequence and structure to catalase and to define the biochemistry and chemistry of their reactions and novel products. Catalase is renowned for its efficient reaction with hydrogen peroxide and its key role in the oxidative defense of all aerobic organisms. The catalase-related relatives studied here are smaller proteins with reaction specificity directly against fatty acid hydroperoxides. The prototypical enzyme of the proposal is an allene oxide synthase, an enzyme that catalyzes a cytochrome P450-type of reaction yet which exhibits distinct sequence homology to catalase.
In Aim 1, the ability to transform the catalase-related AOS and the P450 type of AOS to monooxygenases will be examined using surrogate oxygen donors to activate the heme, with stopped flow spectral recording to detect short-lived intermediates as well as detailed product analysis to define the substrate-enzyme interaction. We also propose to change the AOS activity of these enzymes by site-direct mutagenesis of active site amino acids, a hypothesis supported by the array of products of CYP74 relatives of the P450 AOS.
Aim 2 will focus on analysis of the catalytic activities of other novel enzyme candidates that have similar sequence characteristics defined as retention of the heme-binding features of a catalase within an unusually short polypeptide for catalases of only ~40kD.
Aim 3 will analyze the mechanism of biosynthesis of a unique bicyclobutane fatty acid made by a catalase-related enzyme from the cyanobacterium Anabaena, and also characterize the structures of its hydrolysis and rearrangement products. This has implications regarding the potential synthesis of bicyclobutanes in other areas of biology. We will also address structural issues pertinent to the cyclization of natural allene oxide diastereomers, which is of fundamental interest in understanding the nature of allene oxide metabolism and the chemistry of cyclopentenone synthesis. The results of this study will provide new insights and a new way of thinking about the enzymatic capabilities of a long-recognized protein family with established roles as a sentinel at the forefront of oxidative defense.

Public Health Relevance

Statement Oxidative stress is a key factor underlying the progression of many diseases ranging from atherosclerosis to diabetes, inflammation, and cancer. By uncovering the details of how different enzymes deal with oxidants and how they relate or differ from each other this project will improve our understanding of oxidative stress, which will ultimately help control it.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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Vanderbilt University Medical Center
Schools of Medicine
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Teder, Tarvi; Boeglin, William E; Brash, Alan R (2017) Oxidation of C18 Hydroxy-Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase-Related Hemoproteins Activated with Iodosylbenzene. Lipids 52:587-597
Hebert, Sebastien P; Cha, Jin K; Brash, Alan R et al. (2016) Investigation into 9(S)-HPODE-derived allene oxide to cyclopentenone cyclization mechanism via diradical oxyallyl intermediates. Org Biomol Chem 14:3544-57
Mashhadi, Zahra; Newcomer, Marcia E; Brash, Alan R (2016) The Thr-His Connection on the Distal Heme of Catalase-Related Hemoproteins: A Hallmark of Reaction with Fatty Acid Hydroperoxides. Chembiochem 17:2000-2006
Teder, Tarvi; Lõhelaid, Helike; Boeglin, William E et al. (2015) A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE. J Biol Chem 290:19823-32
Mashhadi, Zahra; Boeglin, William E; Brash, Alan R (2015) Robust inhibitory effects of conjugated linolenic acids on a cyclooxygenase-related linoleate 10S-dioxygenase: Comparison with COX-1 and COX-2. Biochim Biophys Acta 1851:1346-52
Mashhadi, Zahra; Boeglin, William E; Brash, Alan R (2014) Inhibitory effects of a novel Val to Thr mutation on the distal heme of human catalase. Biochimie 106:180-3
Brash, Alan R; Niraula, Narayan P; Boeglin, William E et al. (2014) An ancient relative of cyclooxygenase in cyanobacteria is a linoleate 10S-dioxygenase that works in tandem with a catalase-related protein with specific 10S-hydroperoxide lyase activity. J Biol Chem 289:13101-11
Teder, Tarvi; Boeglin, William E; Brash, Alan R (2014) Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction. J Lipid Res 55:2587-96
Brash, Alan R; Boeglin, William E; Stec, Donald F et al. (2013) Isolation and characterization of two geometric allene oxide isomers synthesized from 9S-hydroperoxylinoleic acid by cytochrome P450 CYP74C3: stereochemical assignment of natural fatty acid allene oxides. J Biol Chem 288:20797-806
Jin, Jing; Boeglin, William E; Cha, Jin K et al. (2012) 8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-?-linolenic acid: a distinctive transformation compared with S-lipoxygenases. J Lipid Res 53:292-9

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