My research focuses on applying crystallographic techniques to aid in understanding flavoenzyme chemistry. Flavoenzymes are proteins containing flavin nucleotide co-enzymes. They catalyze a variety of biologically important reactions using various chemical mechanisms. Factors affecting the catalytic reaction and the reactivity of the flavin are strongly dependent on the interactions between the flavin and the protein environment surrounding it. It is clear that the protein structure in the region of the active site plays a key role in the reactions carried out by these enzymes. In this proposal I aim to use a crystallography to address the question of how the redox properties of different flavoenzymes are related to their catalytic function and to structure of the protein in the active site. From structural interpretations, these relationships will be further probed and modified by genetic engineering and the effect of specific changes at the active changes at the active site both on the thermodynamics and the rates of electron transfer will be assessed. By combining redox data with structural data a structure-function redox potential correlation will be determined which will give insight into the workings of this fascinating group of enzymes. Three different proteins have been chosen for structural studies, 2 oxidases: cholesterol oxidase and L-amino acid oxidase, and one monooxygenase: tryptophan 2-monooxygenase. In all cases the first step in the reaction involves C-H bond cleavage of the substrate and transfer of a hydrogen and 2 electrons to the flavin cofactor. However, subsequent steps in the reaction vary amongst these enzymes. Thus, although these 3 enzymes all undergo a C-H bond cleavage, the chemical nature of the substrate dictates a different mechanism for this cleavage reaction. Crystallographic studies of these enzymes will address the question of how the protein structure facilitates these different mechanisms. The structure will enable us to study the different features which contribute to the stability of the flavin radical formed upon C-H bond cleavage.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063262-02
Application #
6343116
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Lewis, Catherine D
Project Start
2000-08-15
Project End
2005-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
2
Fiscal Year
2001
Total Cost
$199,468
Indirect Cost
Name
University of California Santa Cruz
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
Chen, Lin; Lyubimov, Artem Y; Brammer, Leighanne et al. (2008) The binding and release of oxygen and hydrogen peroxide are directed by a hydrophobic tunnel in cholesterol oxidase. Biochemistry 47:5368-77
Lyubimov, Artem Y; Heard, Kathryn; Tang, Hui et al. (2007) Distortion of flavin geometry is linked to ligand binding in cholesterol oxidase. Protein Sci 16:2647-56
Lim, Louis; Molla, Gianluca; Guinn, Nicole et al. (2006) Structural and kinetic analyses of the H121A mutant of cholesterol oxidase. Biochem J 400:13-22
Moustafa, Ibrahim M; Foster, Scott; Lyubimov, Artem Y et al. (2006) Crystal structure of LAAO from Calloselasma rhodostoma with an L-phenylalanine substrate: insights into structure and mechanism. J Mol Biol 364:991-1002
Lario, Paula I; Vrielink, Alice (2003) Atomic resolution density maps reveal secondary structure dependent differences in electronic distribution. J Am Chem Soc 125:12787-94
Vrielink, Alice; Sampson, Nicole (2003) Sub-Angstrom resolution enzyme X-ray structures: is seeing believing? Curr Opin Struct Biol 13:709-15
Lario, Paula I; Sampson, Nicole; Vrielink, Alice (2003) Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity. J Mol Biol 326:1635-50
Yin, Y; Sampson, N S; Vrielink, A et al. (2001) The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase. Biochemistry 40:13779-87