Characterisation of Potential Antimicrobial Targets in Bacillus spp. I. Aminotransferases and Methionine Regeneration in Bacillus subtilis
Abstract
The aminotransferases involved in the final step of methionine recycling from methylthioadenosine have been examined in the gram-positive bacterium Bacillits subtilis. Homogenates of this bacterium were able to convert ketomethiobutyrate to methionine, utilising leucine, isoleucine, valine, phenylalanine, tyrosine, and alanine as preferred amino donors. Unlike other organisms examined to date in this context, B. subtilis was found to contain no aspartate aminotransferase or tyrosine aminotransferase sequences with structural homology to subfamily Ia aminotransferases. Instead, in B. subtills, the six putative homologues of aspartate aminotransferase were found to be members of the If subfamily. Five of these six sequences were cloned and expressed, with only the ykrV gene product capable of producing methionine from ketomethiobutyrate. However, this enzyme only catalysed the reaction using glutamine as an amino donor. Two putative branched-chain amino acid aminotransferases from family III were also cloned and expressed, and both were found to produce methionine from ketomethiobutyrate using branched-chain and aromatic amino acids. Of these two enzymes, the ybgE gene product was the most active and had kinetic constants consistent with it being the enzyme responsible for the majority of methionine regeneration in this organism. The ybgE gene product could be inhibited uncompetitively by the aminooxy compound canaline, with a Ki of 48 muM. In addition, canaline inhibited the in vitro growth of B. subtilis in minimal medium with an IC50 of 37 muM and an MIC of 500 muM. Growth inhibition in nutrient broth was negligible.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2002
- Accession Number
- ADA405866
Entities
People
- Bradley J. Berger
- Marvin H. Knodel