Purification and some properties of carboxynorspermidine synthase participating in a novel biosynthetic pathway for norspermidine in Vibrio alginolyticus

Carboxynorspermidine synthase, mediates the nicotinamide-nucleotide-linked reduction of the Schiff base H₂N(CH₂)₃N=CHCH₂CH(NH₂)COOH. This is formed from L-aspartic,β-semialdehyde (ASA) and 1,3-diaminopropane (DAP) and is reduced to carboxynorspermidine (H₂N(CH₂)₃NH(CH₂)₂CH(NH₂)COOH), an intermediate in the novel pathway for norspermidine (NSPD) biosynthesis. The enzyme was purified to apparent homogeneity from Vibrio alginolyticus and characterized. The overall purification was about 1800-fold over the crude extract, with a yield of 33%. The enzyme displayed an apparent M(r) of 93500 ± 1000 by gel filtration and 45100 ± 500 by SDS-PAGE, indicating that the native form is probably composed of two subunits of similar size. The specific activity of the purified enzyme was 31.0 μmol carboxynorspermidine produced min^-1(mg protein)^-1. The enzyme was activated by dithiothreitol, and inhibited by SH-reactive compounds. The pH and temperature optima were 7.25-7.5 and 37°C, respectively. The K(m) value for the Schiff base was 4.68 mM, measured by varying the ASA concentration while keeping the DAP concentration constant. Putrescine was slightly active as a substrate, forming carboxyspermidine (at about 7% of the rate of DAP), but ethylenediamine, cadaverine and DASA were inert. The K(m) value for NADPH was 1.51 mM. NADH was a much poorer cofactor than NADPH. When V. alginolyticus was grown in the presence of 5 mM-NSPD, the specific activity of this enzyme was reduced by ~70%. NSPD also repressed two other enzymes responsible for its biosynthesis, 2,4-diaminobutyrate decarboxylase and carboxynorspermidine decarboxylase.