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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National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Harvard University
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Yeh, Ellen; Cole, Lindsay J; Barr, Eric W et al. (2006) Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH. Biochemistry 45:7904-12
Liu, Fei; Garneau, Sylvie; Walsh, Christopher T (2004) Hybrid nonribosomal peptide-polyketide interfaces in epothilone biosynthesis: minimal requirements at N and C termini of EpoB for elongation. Chem Biol 11:1533-42
Lessard, I A; Walsh, C T (1999) Mutational analysis of active-site residues of the enterococcal D-ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-ala-D-Ala ligase and D-ala-D-Ala carboxypeptidase VanY. Chem Biol 6:177-87
Lessard, I A; Walsh, C T (1999) VanX, a bacterial D-alanyl-D-alanine dipeptidase: resistance, immunity, or survival function? Proc Natl Acad Sci U S A 96:11028-32
Stoll, V S; Simpson, S J; Krauth-Siegel, R L et al. (1997) Glutathione reductase turned into trypanothione reductase: structural analysis of an engineered change in substrate specificity. Biochemistry 36:6437-47
Rennex, D; Cummings, R T; Pickett, M et al. (1993) Role of tyrosine residues in Hg(II) detoxification by mercuric reductase from Bacillus sp. strain RC607. Biochemistry 32:7475-8
Smith, K; Nadeau, K; Bradley, M et al. (1992) Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata. Protein Sci 1:874-83
Moore, M J; Miller, S M; Walsh, C T (1992) C-terminal cysteines of Tn501 mercuric ion reductase. Biochemistry 31:1677-85
Leichus, B N; Bradley, M; Nadeau, K et al. (1992) Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase. Biochemistry 31:6414-20
Walsh, C; Bradley, M; Nadeau, K (1992) Molecular studies on trypanothione reductase: an antiparasitic target enzyme. Curr Top Cell Regul 33:409-17

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