The research supported by this grant will focus on structure-function relationships of flavoprotein monooxygenases using biochemical, biophysical and protein engineering methods. A major goal is to develop an understanding in molecular terms of how the different families of flavoprotein monooxygenases can all function through the formation of common oxygen intermediates, the C4a-peroxyflavins, but nevertheless have very diverse substrate specificities and mechanisms of regulation. Because the flavin provides a sensitive built-in spectroscopic reporter group for monitoring individual stages of the reactions, a very detailed examination of the individual reactions, including protein dynamic events, is possible. It is planned to study in detail four main classes of such enzymes: 1) aromatic hydroxylases, which appear to function using the flavin hydroperoxide as a nucleophile; 2) cyclohexanone monooxygenase, an enzyme incorporating an oxygen atom into its substrate by a Baeyer-Villiger reaction, using the flavin C4a peroxide anion as a nucleophile; 3) broad substrate-tolerant enzymes capable of oxygenating a diverse array of nitrogen-and sulfur-compounds; 4) unusual 2-protein component enzymes from bacterial sources that carry out the hydroxylation of p-hydroxyphenylacetate. These enzymes are widely distributed in nature and play important roles in xenobiotic metabolism in animals, biosynthesis of plant hormones and bioremediation processes by soil bacteria. They also offer the possibility of synthesis of chiral compounds that could be useful in drug development. Extensive use will be made of site-directed mutagenesis, based on structural information already available for p-hydroxybenzoate hydroxylase and phenol hydroxylase, and determination of the structure of cyclohexanone monooxygenase is also planned. The reaction mechanism of each enzyme will be explored by rapid reaction techniques, including stopped-flow fluorescence and absorbance spectroscopy, by NMR of isotopic ally labeled protein, and by solid state NMR techniques. Extensive use will also be made of artificial flavins introduced into the enzymes in place of the natural flavins, as probes both of active site structure and mechanism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM064711-01
Application #
6422377
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
2002-02-01
Project End
2006-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
1
Fiscal Year
2002
Total Cost
$381,061
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Nijvipakul, Sarayut; Ballou, David P; Chaiyen, Pimchai (2010) Reduction kinetics of a flavin oxidoreductase LuxG from Photobacterium leiognathi (TH1): half-sites reactivity. Biochemistry 49:9241-8
Chakraborty, Sumita; Ortiz-Maldonado, Mariliz; Entsch, Barrie et al. (2010) Studies on the mechanism of p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa: a system composed of a small flavin reductase and a large flavin-dependent oxygenase. Biochemistry 49:372-85
Nijvipakul, Sarayut; Wongratana, Janewit; Suadee, Chutintorn et al. (2008) LuxG is a functioning flavin reductase for bacterial luminescence. J Bacteriol 190:1531-8
Valton, Julien; Mathevon, Carole; Fontecave, Marc et al. (2008) Mechanism and regulation of the Two-component FMN-dependent monooxygenase ActVA-ActVB from Streptomyces coelicolor. J Biol Chem 283:10287-96
Ballou, David P (2007) Crystallography gets the jump on the enzymologists. Proc Natl Acad Sci U S A 104:15587-8
Suadee, Chutintorn; Nijvipakul, Sarayut; Svasti, Jisnuson et al. (2007) Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues. J Biochem 142:539-52
Sucharitakul, Jeerus; Phongsak, Thanawat; Entsch, Barrie et al. (2007) Kinetics of a two-component p-hydroxyphenylacetate hydroxylase explain how reduced flavin is transferred from the reductase to the oxygenase. Biochemistry 46:8611-23
Yeh, Ellen; Cole, Lindsay J; Barr, Eric W et al. (2006) Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH. Biochemistry 45:7904-12
Sucharitakul, Jeerus; Chaiyen, Pimchai; Entsch, Barrie et al. (2006) Kinetic mechanisms of the oxygenase from a two-component enzyme, p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii. J Biol Chem 281:17044-53
Cole, Lindsay J; Entsch, Barrie; Ortiz-Maldonado, Mariliz et al. (2005) Properties of p-hydroxybenzoate hydroxylase when stabilized in its open conformation. Biochemistry 44:14807-17

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