Branched-chain amino acid aminotransferase and methionine formation in Mycobacterium tuberculosis.
BACKGROUND
Tuberculosis remains a major world-wide health threat which demands the discovery and characterisation of new drug targets in order to develop future antimycobacterials. The regeneration of methionine consumed during polyamine biosynthesis is an important pathway present in many microorganisms. The final step of this pathway, the conversion of ketomethiobutyrate to methionine, can be performed by aspartate, tyrosine, or branched-chain amino acid aminotransferases depending on the particular species examined.


RESULTS
The gene encoding for branched-chain amino acid aminotransferase in Mycobacterium tuberculosis H37Rv has been cloned, expressed, and characterised. The enzyme was found to be a member of the aminotransferase IIIa subfamily, and closely related to the corresponding aminotransferase in Bacillus subtilis, but not to that found in B. anthracis or B. cereus. The amino donor preference for the formation of methionine from ketomethiobutyrate was for isoleucine, leucine, valine, glutamate, and phenylalanine. The enzyme catalysed branched-chain amino acid and ketomethiobutyrate transamination with a Km of 1.77 - 7.44 mM and a Vmax of 2.17 - 5.70 micromol/min/mg protein, and transamination of ketoglutarate with a Km of 5.79 - 6.95 mM and a Vmax of 11.82 - 14.35 micromol/min/mg protein. Aminooxy compounds were examined as potential enzyme inhibitors, with O-benzylhydroxylamine, O-t-butylhydroxylamine, carboxymethoxylamine, and O-allylhydroxylamine yielding mixed-type inhibition with Ki values of 8.20 - 21.61 microM. These same compounds were examined as antimycobacterial agents against M. tuberculosis and a lower biohazard M. marinum model system, and were found to completely prevent cell growth. O-Allylhydroxylamine was the most effective growth inhibitor with an MIC of 78 microM against M. marinum and one of 156 microM against M. tuberculosis.


CONCLUSIONS
Methionine formation from ketomethiobutyrate is catalysed by a branched-chain amino acid aminotransferase in M. tuberculosis. This enzyme can be inhibited by selected aminooxy compounds, which also have effectiveness in preventing cell growth in culture. These compounds represent a starting point for the synthesis of branched-chain aminotransferase inhibitors with higher activity and lower toxicity.
Chemical & Biological Defence Section, Defence R&D Canada - Suffield, Box 4000 Station Main, Medicine Hat, Alberta, T1A 8K6, Canada. esvenos@interchange.ubc.ca <esvenos@interchange.ubc.ca>
Erik S
ES
Venos
Marvin H
MH
Knodel
Cynthia L
CL
Radford
Bradley J
BJ
Berger
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D010802:Phylogeny; D016415:Sequence Alignment; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D008715:Methionine; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase