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 beta-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 Mr 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 mumol 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 degrees C, respectively. The Km 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 D-ASA were inert. The Km 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 approximately 70%. NSPD also repressed two other enzymes responsible for its biosynthesis, 2,4-diaminobutyrate decarboxylase and carboxynorspermidine decarboxylase.
Nakao
H
H
Faculty of Pharmaceutical Sciences, Okayama University Tsushima, Japan.
Shinoda
S
S
Yamamoto
S
S
D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013095:Spermidine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000378:metabolism
D009249:NADP; C006760:norspermidine; D000587:Oxidoreductases Acting on CH-NH Group Donors; C071853:carboxynorspermidine synthase; D004229:Dithiothreitol; D013095:Spermidine