CLONING, EXPRESSION, SEQUENCE-ANALYSIS, AND SITE-DIRECTED MUTAGENESIS OF THE TN5306-ENCODED N-5-(CARBOXYETHYL)ORNITHINE SYNTHASE FROM LACTOCOCCUS-LACTIS K1

The gene (ceo) encoding N-5-(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 N-5-(1-carboxypropyl)-L-ornithine and N-6-(1-carboxypropyl)-L-lysine. Comparison of the deduced amino acid sequence of N-5-(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, N-5-(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 N-5-(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 similar to 80 residues has significant similarity to alanine dehydrogenase. Substitution of arginine 15 of N-5-(1-carboxyethyl)-L-ornithine synthase by lysine resulted in loss of enzyme activity.