A wide range of biochemical functions are linked to the actions of monooxygenases which activate the molecular oxygen for the insertion of one oxygen atom into a variety of organic substrates. Flavoprotein hydroxylases, a sub-family of monooxygenases, are important in detoxification, drug activation or inactivation, biosynthesis of antitumor agent valanimycin, biodegradation of naturally occurring and man-made organic compounds, desulfurization of fossil fuel, bioluminescence, and other processes. The longterm goal of this project is to acquire an integrated understanding of the structures and mechanisms of flavoprotein hydroxylases. During the current funding period, research efforts began to focus on luciferase and the functionally linked flavin reductases from luminous bacteria. The same research focus will be maintained for the next grant period. The main specific aim is to investigate the mechanisms of reduced flavin transfer and physical interactions between flavin reductases and luciferase. In addition, we will map the NADPH site and characterize the function of a mobile loop of the flavin reductase FRP, and to further elucidate the mechanism and structure-function relationships of luciferase. The necessary reduced flavin substrate for luciferase is believed to be supplied in vivo by flavin redutases. We now know that an increasing number of enzymes also require flavin reductases to supply reduced flavin for their activities. However, the mechanisms of reduced flavin transfer are not well understood for any system. Our proposed investigation on flavin reductases with respect to their structures and modes of interaction with and reduced flavin transfer to luciferase is, to our knowledge, the first comprehensive study on reduced flavin transfer mechanisms. Thus, the proposed study should be of interest to diverse areas in biological and biomedical sciences. Luciferase, due to its extremely slow turnover and the unique ability to emit light, offers special challenges and advantages for enzymological investigations. Certain activated oxygen intermediates common to all known flavo-hydroxylases can be uniquely studied in isolated forms using the luciferase system. Moreover, luciferase and its genes provide one of the most versatile reporter systems for basic, biomedical and biotechnological research. The proposed investigations on luciferase will impact on fields beyond the immediate area of flavoprotein hydroxylases.
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