This research program focuses on a detailed investigation into the enzymology of choline oxidase (CHO), a flavin-dependent enzyme belonging to a highly diversified superfamily of flavoenzymes that catalyze the oxidation of alcohols, the Glucose-Methanol-Choline (GMC) oxidoreductase superfamily. CHO catalyzes the four-electron oxidation of choline to glycine betaine through two sequential flavin-linked hydride transfer reactions from choline and the ensuing betaine aldehyde intermediate to molecular oxygen. The specific goals of the research program are to: i) Establish the importance of limited independent substrate motion with respect to catalytic groups, so that the only dynamical movements permitted in the activated enzyme-substrate complex are those that contribute to efficient catalysis. ii) Identify the active site amino acid residue that activates the alcohol substrate by removal of the hydroxyl proton. iii) Establish that oxygen activation for reaction with the reduced flavin is provided by the positively charged headgroup of the enzyme-bound aldehyde intermediate. The experimental approach will combine kinetic and biochemical analyses, substrate deuterium, solvent, and 18O-kinetic isotope effects, as well as mutagenesis and crystallography. The results that will be gathered from this project will significantly advance the knowledge and understanding of the enzymology of CHO and, to a broader extent, of the structural and functional factors that are required for efficient catalysis and oxygen reactivity in flavin-dependent enzymes.
Broader Impacts The research project will foster the integration of exciting research with the education of minority students, enabling them to develop skills and knowledge in mechanistic enzymology, biochemistry, protein chemistry, and molecular biology. Knowledge and skills in these disciplines are becoming increasingly indispensable in the post-genome era in order to shorten the existing gap between scientific knowledge at the genomic level, where a wealth of information is already available, and post-genomic level, where knowledge is lagging behind. Knowledge and level of training that will be acquired by students through the achievement of the aims of the project will be not only of significance for the understanding of the enzyme under study, but will be an invaluable asset for the future studies of other enzymes and proteins. This work will be pursued in the context of Dr. Gadda's educational activities. He will continue and expand his outreach contribution in the NSF Center for Workshop in the Chemical Sciences and in an interactive two-way television network seminar series that is broadcast live to colleges in Georgia.
The enzymatic oxidation of alcohols is a reaction that is central to a number of biochemical pathways. It is catalyzed by a large number of enzymes that utilize either zinc, pyrroloquinoline quinone or flavins as cofactors for the reaction. In this research project, we have investigated the structure-function relationships of choline oxidase, a flavin-dependent enzyme that catalyzes the four-electron oxidation of choline to glycine betaine through formation of an aldehyde intermediate. Molecular oxygen acts as electron acceptor in both the oxidation reactions catalyzed by the enzyme. Three major outcomes emerged from the project. First, the importance of limited independent movement of the alcohol substrate with respect to the catalytic groups and the flavin cofactor in the active site of the enzyme for efficient catalysis was established through biophysical, structural and mechanistic approaches. Second, a detailed mapping of the roles for catalysis, substrate binding or substrate positioning of several amino acid residues in the active site of the enzyme was carried out using mutagenic, biochemical, structural and mechanistic approaches. Third, it was established that oxygen activation for reaction with the enzyme-bound reduced flavin is provided almost entirely by the positive charge harbored on the enzymatic product of the reaction rather than on the enzyme. These project outcomes have thus significantly advanced the knowledge and understanding of the structural and functional determinants that are required for efficient alcohol oxidation and oxygen reactivity in choline oxidase and, more in general, in flavin-dependent enzymes. The scientific outcomes of this research have been published in more than thirty peer-reviewed publications in scientific journals. The PI and his students have presented the scientific outcomes of the research with more than seventy-five poster presentations at international, national, regional and local conferences. The PI presented more than twenty-nine invited talks at conferences and other Universities in the US and abroad. Thirty-two among undergraduate, graduate and post-graduate students have been involved to different extents in the project. These students have been trained in biochemistry, mechanistic enzymology, molecular biology and protein chemistry. Five undergraduate students have appeared as co-authors in peer-reviewed scientific publications with the PI, whereas graduate students and postdoctoral research associates have authored with the PI twenty-seven peer-reviewed scientific articles. Undergraduate and graduate students have presented several invited talks, most notably at the 12th Annual SAEOPP/UTK McNair National Scholars Research Conference, Knoxville, TN (2006), Isotopes 2009, Cluj-Napioca, Romania, Southeast Enzyme Conference, Atlanta (2011) and Gordon Research Seminar on Isotopes in Biological and Chemical Sciences, Galveston, TX (2012). Five graduate students have earned their PhD degrees with the PI while working on this project and went on to postdoctoral or lecturer positions at various US Universities and at the Center for Disease Control and Prevention. Two students graduated with a Master of Science degree and had been employed as research scientists in the industry and at the Center for Disease Control and Prevention. Several of the undergraduate students associated with the project have graduated and furthered their education by enrolling in graduate programs in Atlanta and across the nation.
