FLAVOENZYME MECHANISMS: REDOX AND NON-REDOX REACTIONS

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DESCRIPTION (applicant's description): The proposed research involves a comprehensive investigation of the relationship of structure to function in monomeric sarcosine oxidase (MSOX) and heterotetrameric sarcosine oxidase (TSOX), bacterial enzymes used in the clinical evaluation of renal function. The overall goal is to gain a deeper understanding of how flavin-containing enzymes catalyze amine oxidation and 1-carbon transfer to tetrahydrofolate, reactions of considerable physiological importance. MSOX and TSOX are members of a major superfamily of amine oxidoreductases that contains a number of clinically important enzymes like monoamine oxidase and sarcosine dehydrogenase, an enzyme defective in patients with sarcosinemia. These amine oxidoreductases all exhibit a requirement for covalent incorporation of flavin (FAD or FMN). MSOX and TSOX catalyze the oxidative demethylation of sarcosine (N-methylglycine) but exhibit notable structural and functional differences. MSOX is a monomeric protein containing covalently bound FAD. TSOX is a multimeric enzyme that contains three coenzymes (FAD, NAD+ and covalently bound FMN) and catalyzes both sarcosine oxidation and synthesis of 5,10-methylenetetrahydrofolate; the beta subunit appears to be the structural homolog of MSOX. The proposed studies build on previous work by the PI Studies with MSOX, guided by high resolution structures (1.3 - 2.0 A) of the free enzyme and its complexes with substrate analogs, have the following principal objectives: Determination of the mechanism of sarcosine oxidation; evaluation of the role of the covalent flavin linkage in catalysis; determination of the mechanism of covalent flavin attachment; evaluation of the factors that modulate flavin redox properties. Studies with TSOX build on our success in obtaining diffraction quality (2.8 A) crystals and have two major objectives: Elucidation of the structural and functional organization of the multiple subunits and coenzymes in this complex bifunctional enzyme; determination of the mechanism for efficient coupling of sarcosine oxidation with 5,10-methylenetetrahydrofolate synthesis, chemistry relevant to the reactions catalyzed by sarcosine dehydrogenase. These studies represent a multidisciplinary approach, involving rapid reaction kinetics, stereochemical analysis, mutagenesis, crystallography, use of mechanism-based inhibitors and magnetic field effects.