. Glutathione is considered a key player in protecting organisms against oxygen toxicity. However, it is absent in mycobacteria which are highly resistant to oxidative stress indicating that they use alternative thiol-protective mechanisms. It has been shown that mycobacteria produce considerable levels of a novel cysteine derivative named mycothiol which is resistant to oxidation. The investigators have shown that M. smegmatis is an order of magnitude more resistant to the thiol-oxidizing agent diamide than E. coli and other mammalian cells. This resistance appears to be due to their ability to produce mycothiol. The goal of this project is elucidate the pathways involved in mycothiol biosynthesis and utilization and to identify potential targets for TB drug development.
The specific aims are designed 1) to develop a chemical synthesis of mycothiol and its components for use in testing the biosynthesis and function of mycothiol, 2) to determine the pathway of mycothiol biosynthesis, 3) to purify the coA cysteine acetyl-transferase activity detected in M. Smegmatis and determine whether it functions in the initial step of mycothiol biosynthesis, 4) to determine the spectrum of enzyme activity utilizing mycothiol substrate, and 5) to obtain mutants blocked in the synthesis of mycothiol and establish their sensitivity to oxidative challenge.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA011393-03
Application #
2732464
Study Section
AIDS and Related Research Study Section 5 (ARRE)
Project Start
1996-07-01
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
2000-06-30
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Newton, Gerald L; Fahey, Robert C (2002) Mycothiol biochemistry. Arch Microbiol 178:388-94
Newton, G L; Av-Gay, Y; Fahey, R C (2000) N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. J Bacteriol 182:6958-63
Newton, G L; Av-Gay, Y; Fahey, R C (2000) A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase. Biochemistry 39:10739-46
Unson, M D; Newton, G L; Arnold, K F et al. (1999) Improved methods for immunoassay of mycothiol. J Clin Microbiol 37:2153-7
Newton, G L; Unson, M D; Anderberg, S J et al. (1999) Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis. Biochem Biophys Res Commun 255:239-44
Anderberg, S J; Newton, G L; Fahey, R C (1998) Mycothiol biosynthesis and metabolism. Cellular levels of potential intermediates in the biosynthesis and degradation of mycothiol in mycobacterium smegmatis. J Biol Chem 273:30391-7
Unson, M D; Newton, G L; Davis, C et al. (1998) An immunoassay for the detection and quantitative determination of mycothiol. J Immunol Methods 214:29-39