Publications

• N(5)-(L-1-carboxyethyl)-L-ornithine:NADP(+) oxidoreductase from Streptococcus lactis. Purification and partial characterization.

  Thompson J.

  N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to fo ... >> More

  N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation. << Less

  J. Biol. Chem. 264:9592-9601(1989) [PubMed] [EuropePMC]

• The gene CBO0515 from Clostridium botulinum strain Hall A encodes the rare enzyme N5-(carboxyethyl) ornithine synthase, EC 1.5.1.24.

  Thompson J., Hill K.K., Smith T.J., Pikis A.

  Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N(5)-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and c ... >> More

  Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N(5)-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum. << Less

  J. Bacteriol. 192:1151-1155(2010) [PubMed] [EuropePMC]