Four problems in flavoenzyme structure and catalytic mechanism are to be studied, reflecting both the diversity of electron transfer chemistry available to flavin coenzymes and the biological range of reactions utilizing vitamin B2 derivatives. 1) We shall continue studies on the mer A gene and its encoded enzyme, mercuric ion reductase, conferring resistance to inorganic mercuric salts to bacteria that carry this and associated genes of the mer operon. Site-directed mutagenesis of sulfhydryl groups will continue to be a focus. 2) Cyclohexanone Oxygenase enables soil bacteria to grow on cycloalkanones as carbon source and is of interest as the best characterized biological Baeyer- Villiger oxygenation catalyst. We have cloned and sequenced the Acinetobacter gene and will continue analysis of structure and catalytic mechanism for oxygen activation and transfer to cosubstrates. 3) Cyclobutane-containing intrachain thymine dimers are the major lesions in UV-damaged DNA, and in several species (prokaryotes and eukaryotes) these can be repaired in a visible-light driven photomonomerization reaction. The photoreactivation enzyme from streptomycetes, blue green alga Anacystis nidulans, and from methanobacteria appear to contain both FAD and the 8-hydroxy-5-deazaflavin factor F420. We will analyze how visible light is utilized in this two coenzyme sequence to photorepair DNA. 4) In kinetoplastid-containing parasites such as trypanosomes and leishmania there is very little glutathione; most glutathione is modified as the N1,8-bis spermidinyl derivative known as trypanothione. We have determined there is no detectable glutathione reductase in these parasites but rather a specific trypanothione reductase which we have purified to homogeneity and characterized as an analogous flavoenzyme. We are in the process of cloning and sequencing the gene from various trypanosomatids and will continue molecular biology and enzymology studies on this enzyme, likely target for antiparasitic agents.
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