Bradley J. Berger

Methionine Regeneration and Aminotransferases in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis

The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in B. subtilis were found to be members of the If subfamily. These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of B. subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in B. subtilis, ybgE and ywaA, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of B. cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms. In both B. cereus and B. anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from B. cereus, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of B. subtilis YbgE and also inhibited growth of B. subtilis and B. cereus in culture.

Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa

Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.

Characterization of the ornithine aminotransferase from Plasmodium falciparum.

The ornithine aminotransferase from Plasmodium falciparum 3D7 was cloned, functionally expressed, and characterized. The gene exists as a single copy in the malarial genome and is located on chromosomes 6/7/8. The deduced amino acid sequence was found to be 85% identical to a similar sequence discovered in Plasmodium yoelii, 82% identical to a partial sequence from Plasmodium vivax, and 42-53% identical to ornithine aminotransferases from other eukaryotes. The enzyme had a very narrow substrate specificity, and could only catalyze the transamination of alpha-ketoglutarate with ornithine or N-acetylornithine, and of glutamate-5-semialdehyde with glutamate and alanine. The aminooxy analogue of ornithine, canaline, was found to inhibit the ornithine aminotransferase uncompetatively with a Ki of 492+/-98 nM. As the enzyme effectively catalyzed both ornithine catabolism and formation, its potential role in ornithine biosynthesis from glutamine, via glutamate, glutamate-5-phosphate, and glutamate-5-semialdehyde, was examined. Over the course of a 3.5 h incubation, P. falciparum converted 34% of exogenous, radiolabeled glutamine to glutamate and 0.68% to ornithine. This low level of conversion suggests that the parasite may have alternative mechanisms for obtaining ornithine for polyamine biosynthesis.

Branched-chain amino acid aminotransferase and methionine formation in Mycobacterium tuberculosis

BackgroundTuberculosis 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.ResultsThe 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 μmol/min/mg protein, and transamination of ketoglutarate with a Km of 5.79 – 6.95 mM and a Vmax of 11.82 – 14.35 μmol/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 μM. 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 μM against M. marinum and one of 156 μM against M. tuberculosis.ConclusionMethionine 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.

Antimalarial activities of aminooxy compounds

ABSTRACT Twenty-three aminooxy compounds have been examined for their ability to inhibit the growth of the malaria parasite Plasmodium falciparum in vitro. Eight of these compounds were found to have 50% inhibitory concentrations less than 10 μM, with the best drugs being canaline (the aminooxy analogue of ornithine) and CGP51905A at 297 ± 23.6 nM and 242 ± 18.8 nM, respectively. Canaline was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found to be synergistic in antimalarial activity.