Cloning, Expression, Sequence Analysis, and Site-directed Mutagenesis of the Tn5306-encoded N5-(Carboxyethyl)ornithine Synthase from Lactococcus lactisK1 *

The gene (ceo) encoding N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) has been isolated from the sucrose-nisin transposon Tn5306of Lactococcus lactisK1, sequenced, and expressed at high level in Escherichia coli. The cloned enzyme has allowed the synthesis of the novel Nω-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 (Mr= 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 βαβ-fold of the nucleotide binding domain of several microbial NADPH-dependent glutamate dehydrogenases. A much longer sequence of ~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.