Characterisation of Potential Antimicrobial Targets for Tuberculosis. I. Methionine adenosyltransferase in Mycbacterium tuberculosis and M. smegmatis

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Authors
  1. Berger, B.J.
  2. Knodel, M.H.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN)
Abstract
Tuberculosis remains a key concern for the Canadian Forces in its overseas deployments. As drug-resistant forms of the disease continue to spread, there is a need to discover and characterize new drug targets in the organism. The enzyme methionine adenosyltransferase (S-adenosylmethionine synthetase), which catalyzes the formation of S-adenosylmethionine from methionine and ATP, has been cloned, expressed, and characterized in Mycobacterium tuberculosis and its common model organism M. smegmatis. The two gene sequences were both 1200 base pairs in length, and 87% identical with respect to the primary amino acid sequence. Both enzymes also completely conserved all amino acid residues previously identified in Escherichia coli by Takusagawa et al. (Journal of Biological Chemistry, 271, p. 136-47, 1996) as playing a role in substrate MG(2+)/K(+) cofactor binding. Both sequences were expressed in E. coli as calmodulin-binding peptide fusion proteins, with the M. tuberculosis enzyme yielding complete, inactive inclusion body formation. Alteration of various expression and refolding conditions did not provide active, soluble enzyme. The M. smegmatis enzyme, however, was expressed as soluble, active protein. This enzyme had a Vmax of 1.3 µmol/min/mg protein and a Kcat of 0.93 s (exp(-1)) for the formation of S-adenosylmethionine, and a Km of 288 µM for methionine and one of 76 µM for ATP. Eleven methionine analogues and 33 purine analogues were screened as inhibitors of the M. sme

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Keywords
Tuberculosis;Mycobacterium tuberculosis;Mycobacterium smegmatis;Methionine adenosyltransferase;S-adenosylmethionine;Inhibition;Azaguanine;Azathioprine
Report Number
DRDC-SUFFIELD-TR-2003-032 — Technical Report
Date of publication
01 Apr 2003
Number of Pages
36
DSTKIM No
CA022801
CANDIS No
519798
Format(s):
Hardcopy;Document Image stored on Optical Disk

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