Cloning, expression, sequence analysis, and site-directed mutagenesis of the Tn5306-encoded N5-(carboxyethyl)ornithine synthase from Lactococcus lactis K1.
The gene (ceo) encoding N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) has been isolated from the sucrose-nisin transposon Tn5306 of Lactococcus lactis K1, sequenced, and expressed at high level in Escherichia coli. The cloned enzyme has allowed the synthesis of the novel N omega-carboxypropyl amino acids N5-(1-carboxypropyl)-L-ornithine and N6-(1-carboxypropyl)-L-lysine. Comparison of the deduced amino acid sequence of N5-(1-carboxyethyl)-L-ornithine synthase (M(r) = 35,323) to the functionally analogous octopine and nopaline synthases from crown gall tumors showed surprisingly little similarity. However, N5-(1-carboxyethyl)-L-ornithine synthase and yeast saccharopine dehydrogenase exhibit homology at their N and C termini, which suggests that these two proteins constitute a distinct branch of the amino acid dehydrogenase superfamily. A centrally located 9-amino acid segment (GSGNVAQGA) in N5-(1-carboxyethyl)-L-ornithine synthase is virtually identical with a sequence present in the beta alpha beta-fold of the nucleotide binding domain of several microbial NADPH-dependent glutamate dehydrogenases. A much longer sequence of approximately 80 residues has significant similarity to alanine dehydrogenase. Substitution of arginine 15 of N5-(1-carboxyethyl)-L-ornithine synthase by lysine resulted in loss of enzyme activity.
Donkersloot
J A
JA
Laboratory of Microbial Ecology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
Thompson
J
J
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000737:chemistry; D013294:Lactococcus lactis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012443:Saccharopine Dehydrogenases / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D005969:Glutamate Dehydrogenase; D012443:Saccharopine Dehydrogenases; C021883:nopaline synthase; C021903:D-octopine dehydrogenase; C064774:N(5)-(carboxyethyl)ornithine synthase