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 M.The amino acid methionine (Met) is required for a number of vital cellular functions, including the initiation of protein synthesis, the methylation of rRNA and xenobiotics, and the biosynthesis of cysteine, phospholipids, and polyamines. This latter function is particularly important in rapidly growing cells, such as most parasites, bacteria, and cancer cells, which synthesize large amounts of polyamines immediately prior to DNA replication (31). The formation of spermidine from putrescine and of spermine from spermidine consumes Met (in...

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An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of α-ketomethiobutyrate to methionine, has been purified fromKlebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, andRhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with α-ketomethiobutyrate and of glutamate with α-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with α-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.

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The molecular mechanism for polyamine-stimulated feedback modification of ornithine decarboxylase isolated from Physarum polycephalum was investigated by using two-dimensional polyacrylamide-gel electrophoresis. Partially purified A-form enzyme was converted into the B-form enzyme by isolated fractions of the Physarum A-B-converting protein, and the substrates and products were subsequently labelled by covalent addition of alpha-difluoro[14C]methylornithine, an enzyme-activated irreversible inhibitor. The active (A-form) and inactive (B-form) states of this enzyme were found to have the same Mr value, 52 000, yet they differed noticeably in their pI values, 5.45 and 5.65 respectively. In further experiments, the use of high-specific-radioactivity [3H]spermidine to stimulate this enzyme modification was shown not to result in the covalent attachment of this polyamine to ornithine decarboxylase. These results demonstrate that the polyamine-induced modification of ornithine decarboxylase in Physarum is not due to any of the mechanisms previously suggested for ornithine decarboxylase inactivation in this and other eukaryotes, namely phosphorylation, covalent polyamine addition or the non-covalent association of a specific low-Mr protein.