This competing continuation application proposes the extension of mechanistic and structural analyses of flavoenzymes that function in key physiological and protective roles in microorganisms. Typical examples of flavoprotein disulfide reductases that serve this function include the sequence-related alkylhydroperoxide reductase, NADH peroxidase, glutathione reductase and the trypanosomal ortholog, trypanothione reductase, found uniquely in parasitic protozoans. With the determination of the genome sequence of the human pathogenic bacterium, Mycobacterium tuberculosis, the identification of putative flavin-containing enzymes using advanced sequence searching methods has become possible. Using these methods, a number of genes have been identified which are likely to encode flavoproteins. The subsequent cloning of these genes, and the expression and purification of the gene products has confirmed putative assignments based on primary sequence considerations. One example of the application of this method includes the identification of the flavin-containing disulfide reductase that catalyzes the reduction of the oxidized form of mycothiol; a recently discovered, structurally unique thiol present at high concentrations in mycobacteria and a limited number of additional bacterial species. It is likely that this enzyme is responsible for maintaining a reducing intracellular environment, and may contribute to the ability of the bacterium to survive the hostile oxidizing environment encountered in the macrophage phagolysosomal compartment. Two other sequence-related flavoenzymes have been identified, and the principal investigator will attempt to define the physiological function of these two proteins, determine their chemical mechanism and attempt to determine their three-dimensional structure. The principal investigator proposes that these latter enzymes play key roles in the oxidative stress management, survival of the organism and propagation of the infection. The experiments described are designed to assess these roles, and if found to be correct, could ultimately lead to the development of specific inhibitors with chemotherapeutic utility.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033449-20
Application #
6699397
Study Section
Biochemistry Study Section (BIO)
Program Officer
Preusch, Peter C
Project Start
1984-04-01
Project End
2006-01-31
Budget Start
2004-02-01
Budget End
2006-01-31
Support Year
20
Fiscal Year
2004
Total Cost
$375,750
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Argyrou, Argyrides; Blanchard, John S (2004) Flavoprotein disulfide reductases: advances in chemistry and function. Prog Nucleic Acid Res Mol Biol 78:89-142
Argyrou, Argyrides; Blanchard, John S (2004) Kinetic and chemical mechanism of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate isomeroreductase. Biochemistry 43:4375-84
Argyrou, Argyrides; Vetting, Matthew W; Blanchard, John S (2004) Characterization of a new member of the flavoprotein disulfide reductase family of enzymes from Mycobacterium tuberculosis. J Biol Chem 279:52694-702
Argyrou, Argyrides; Sun, Guangxing; Palfey, Bruce A et al. (2003) Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level. Biochemistry 42:2218-28
Vetting, Matthew W; Roderick, Steven L; Yu, Michael et al. (2003) Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases. Protein Sci 12:1954-9
Argyrou, Argyrides; Blanchard, John S; Palfey, Bruce A (2002) The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis. Biochemistry 41:14580-90
Magnet, S; Lambert, T; Courvalin, P et al. (2001) Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase. Biochemistry 40:3700-9
Argyrou, A; Blanchard, J S (2001) Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes. Biochemistry 40:11353-63
Patel, M P; Blanchard, J S (2001) Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects. Biochemistry 40:5119-26
Barlow, J N; Blanchard, J S (2000) Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase. Carbohydr Res 328:473-80

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