Our long term objectives are to elucidate the mechanism of oxidation- reduction reactions catalyzed by flavoenzymes and to evaluate the role of flavin in enzymes where catalysis does not involve a net oxidation- reduction reaction. Corynebacterial sarcosine oxidase, an enzyme useful in clinical diagnosis of kidney function, is a heterotetrameric protein ((alpha beta delta gamma) containing covalent (beta subunit) and noncovalent flavins, a single binding site for sarcosine and two sites for another substrate, tetrahydrofolate. The (beta subunit exhibits sequence homology with various monomeric amine oxidoreductases containing only covalent flavin. To determine whether this new family of flavoenzymes also includes mammalian sarcosine dehydrogenase, we plan to sequence the genes for pig and human sarcosine dehydrogenase. Information on the human gene may be useful in elucidating the molecular basis of sarcosinemia, an autosomal recessive disease characterized by a deficiency of sarcosine dehydrogenase, and high levels of sarcosine in plasma and urine, and possibly associated with neurological and other problems. The functional significance of the complex quaternary structure of corynebacterial sarcosine oxidase will be probed in studies to locate binding sites for various ligands and to determine whether all subunits are essential for sarcosine oxidation. We also seek to elucidate the mechanism of electron transfer from sarcosine to the noncovalent flavin, the kinetics and thermodynamics of interflavin electron transfer, and the biosynthesis and catalytic significance of the covalent flavin linkage. We plan to identify the cysteine residue which forms a reversible complex with the covalent flavin in what appears to represent a novel mechanism of enzyme regulation. The mechanism of human sarcosine dehydrogenase, including its interaction with tetrahydrofolate and the role and biosynthesis of the covalent flavin linkage, will be investigated and compared with the more complex corynebacterial enzyme. Another project involves DNA photolyases from Escherichia coil and Saccharomyces cerevisiae. These enzymes contain pterin and reduced flavin chromophores which enable them to use visible light to repair pyrimidine dimers, the major damage caused by exposure of DNA to ultraviolet light. If left unrepaired, this DNA damage can cause mutations, cancer and cell death. The pterin chromophore binds to a N-terminal domain and acts as an antenna, harvesting light energy which is then transferred to the reduced flavin. The reduced flavin binds to a C-terminal domain and its excited singlet state is the species that directly interacts with substrate and initiates DNA repair. We seek to determine the structure of yeast photolyase and to investigate the specificity and importance of interdomain interactions by creating chimeric enzymes. We seek to characterize the flavin environment and any perturbations induced by substrate. We seek to investigate the mechanism of dimer repair in studies involving time-resolved spectroscopic techniques.
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