The many flavoproteins that catalyze oxidation of carbon-nitrogen and carbon-oxygen bonds by transferring a hydride equivalent to the flavin play critical roles throughout metabolism. A number of these enzymes are targets for treatment of major diseases, so that better understanding of their mechanisms could provide insight for development of therapeutic agents. These enzymes also belong to multiple structural families;it is unclear if this reflects multiple strategies to catalyze essentially identical reactions or is a example of convergent evolution. We have interpreted previous studies of alcohol, keto acid, and amino acid oxidizing enzymes as consistent with a common hydride transfer mechanism for all, with CN and CO bond oxidizing enzymes differing in the need for an active site base in the latter. We now propose to carry out experiments to determine if enzymes that oxidize simple amines and polyamines utilize this same mechanism despite differences in protein and substrate structure. We will determine the mechanism of L- hydroxy-nicotine oxidase, an enzyme in the monoamine oxidase structural family that is proposed to catalyze the oxidation of a carbon-carbon bond rather than a carbon-nitrogen bond. We will continue our studies of polyamine oxidases, enzymes that oxidize the same substrates with different substrate specificities. 15N and 13C kinetic isotope effects will be used to determine the mechanism of amine oxidation, and analysis of the effects of site-directed mutations combined with crystallography will be used to determine the structural basis for the different specificities. We will measure 13C isotope effects for two structural classes of flavin amine oxidases to better define their transition state structures and to provide baseline values for other structural familis. We will determine if members of the trimethylamine dehydrogenase structural family use the same mechanism for amine oxidation as other structural families of amine-oxidizing enzymes. The results of these experiments will test our hypothesis that the different structural families of amine oxidizing flavoenzymes are examples of convergent evolution on a common catalytic mechanism, potentially providing a unifying mechanism for a structurally divergent group of enzymes. Finally, we will initiate studies of the oxidative reactions of flavin-dependent amine oxidases, to probe for intermediates in the reaction and for oxygen-binding sites.

Public Health Relevance

Flavoproteins oxidation that catalyze of amines play central roles in metabolism and are thus frequent targets of drugs. The experiments proposed here will provide fundamental insights into the relationship of protein structure to mechanism and binding specificity for this large group of enzymes. The results will be useful in the design of specific drugs targeting individual enzymes and of new enzymes that catalyze synthetically useful reactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM058698-14A1
Application #
8758342
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
1999-01-01
Project End
2018-03-31
Budget Start
2014-07-10
Budget End
2015-03-31
Support Year
14
Fiscal Year
2014
Total Cost
$299,000
Indirect Cost
$99,000
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Fitzpatrick, Paul F (2017) Nitroalkane oxidase: Structure and mechanism. Arch Biochem Biophys 632:41-46
Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan et al. (2017) Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues. Biochemistry 56:869-875
Trimmer, Elizabeth E; Wanninayake, Udayanga S; Fitzpatrick, Paul F (2017) Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase. Biochemistry 56:2024-2030
Tormos, José R; Suarez, Marina B; Fitzpatrick, Paul F (2016) 13C kinetic isotope effects on the reaction of a flavin amine oxidase determined from whole molecule isotope effects. Arch Biochem Biophys 612:115-119
Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan et al. (2016) Mechanism of the Flavoprotein L-Hydroxynicotine Oxidase: Kinetic Mechanism, Substrate Specificity, Reaction Product, and Roles of Active-Site Residues. Biochemistry 55:697-703
Fitzpatrick, Paul F (2015) Combining solvent isotope effects with substrate isotope effects in mechanistic studies of alcohol and amine oxidation by enzymes. Biochim Biophys Acta 1854:1746-55
Roberts, Kenneth M; Tormos, José R; Fitzpatrick, Paul F (2014) Characterization of unstable products of flavin- and pterin-dependent enzymes by continuous-flow mass spectrometry. Biochemistry 53:2672-9
Gadda, Giovanni; Fitzpatrick, Paul F (2013) Solvent isotope and viscosity effects on the steady-state kinetics of the flavoprotein nitroalkane oxidase. FEBS Lett 587:2785-9
Gaweska, Helena M; Taylor, Alexander B; Hart, P John et al. (2013) Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. Biochemistry 52:2620-6
Gaweska, Helena M; Roberts, Kenneth M; Fitzpatrick, Paul F (2012) Isotope effects suggest a stepwise mechanism for berberine bridge enzyme. Biochemistry 51:7342-7

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