Characterization of Potential Antimicrobial Targets in Bacillus spp. II. Branched-Chain Aminotransferase and Methionine Regeneration in B. cereus and B. anthracis
Abstract
The final step of methionine recycling from methylthioadenosine has been examined in the gram-positive bacteria Bacillus cereus and B. anthracis. Subcellular homogenates were able to convert ketomethiobutyrate to methionine using leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, and alanine as amino donors. Four putative family III aminotransferases, two with homology to branched-chain amino acid aminotransferases and two with homology to D- amino acid aminotransferases, were cloned from B. cereus. The two branched-chain aminotransferases were found to have a low sequence identity with the corresponding enzymes from B. subtilis, indicative of membership of a different subfamily. After expression of the B. cereus enzymes in Escherichia coli and subsequent purification, one branched chain aminotransferase, designated Bc-BCAT2, was found to catalyse methionine regeneration using leucine, isoleucine, valine, phenylalanine, tryosine, and tryptophan as amino donors. The homologue of Bc-BCAT2 was cloned from B. anthracis and designated Ba-BCAT2. Expression of the recombinant enzyme in E. coli and subsequent purification yielded a protein which catalysed methionine regeneration using branched-chain and aromatic amino acids as the amino donors. Kinetic analysis showed that the Km and Vmax values for the enzymes were similar for leucine, valine, and isoleucine as amino donors and ketomethiobutyrate and ketoglutarate as amino acceptors with the Km = 0.41 - 4.34 mM and the Vmax= 0.13 - 1.44 nmol/min/mg protein. Therefore, in both B. cereus and B. a,anthracis, BCAT2 would appear to be the primary catalyst of methionine production from ketomethiobutyrate. The aminotransferase inhibitor canaline was found to inhibit the growth of B. cereus with an IC50 of 35 %micronM in minimal medium and 760 micronM in nutrient broth. The activity in minimal medium was only marginally antagonised by the addition of exogenous methionine or protein.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2002
- Accession Number
- ADA408982
Entities
People
- B. J. Berger
- G. Chan
- M. H. Knodel
- S. English