QUINOENZYMES: BIOGENESIS, STRUCTURE AND FUNCTION

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Quinoproteins constitute a new class of catalysts in which their cofactor is derived from a pre-existing amino acid side chain. Cofactors identified thus far are tryptophan tryptophanylquinone, restricted to prokaryotes, 2,4,5-trihydroxyphenylalanine quinone (TPQ), found to be ubiquitous and lysine tyrosylquinone, restricted to mammalian systems. The latter two cofactors were discovered in the laboratory of the P.I. The family of TPQ enzymes has been demonstrated to catalyze cofactor formation in an auto-catalytic manner, raising the question of a how a single protein structure supports the dual functionalities of cofactor biogenesis and catalytic turnover. The yeast amine oxidase from Hansenula polymorpha (HPAO) is our experimental system of choice, since this protein has been expressed in both S. cerevisiae (for studies of catalytic turnover) and in E. coli (for studies of biogenesis). A crystal structure of the wild type enzyme was solved during the previous granting period and studies of numerous mutant forms will be pursued during the projected period. A series of kinetic and structural studies are planned using both WT and mutant enzymes, with the goal of elucidating the nature of chemical intermediates and rate limiting steps in biogenesis. With regard to catalytic turnover, a new mechanism proposed for the oxidative half reaction will be tested via mutagenesis experiments. Several structural motifs have been identified that involve interaction between residues on opposite subunits of the catalytic dimer; the role of these motifs in biogenesis and turnover will be examined. The physiological function of lysyl oxidase is well defined, whereas that of TPQ-containing molecules in higher eukaryotes remains unknown. Additional studies will involve (i) the investigation of the physiological role of the membrane associated TPQ enzymes in higher eukaryotes, (ii) structure function studies of lysyl oxidase, for comparison to HPAO, and (iii) studies of model compounds for both known and potentially new quinocofactors.