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Literature summary extracted from

  • Nakao, H.; Shinoda, S.; Yamamoto, S.
    Purification and some properties of carboxynorspermidine synthase participating in a novel biosynthetic pathway for norspermidine inVibrio alginolyticus (1991), J. Gen. Microbiol., 137, 1737-1742.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.4.1.B4 DTT without addition 7% of the maximum activity detected in the purified enzyme, 14fold activiation is observed at 20 mM Vibrio alginolyticus
1.4.1.B4 additional information 1 mM EDTA shows no stimulatory effect on enzyme activity Vibrio alginolyticus
1.5.1.43 dithiothreitol about 14fold increase of activity at 20 mM. Without addition of dithiothreitol to the assay mixture, only 7% of the maximum activity is detected in the purified enzyme Vibrio alginolyticus
1.5.1.43 additional information EDTA (1 mM) shows no significant stimulatory effect on enzyme activity Vibrio alginolyticus

General Stability

EC Number General Stability Organism
1.4.1.B4 no significant stabilization by addition of NADPH. 1 mM DTT has a protective effect on the enzyme during purification and storage. Freezing and thawing results in almost complete loss of activity Vibrio alginolyticus

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.4.1.B4 1,2-diaminopropane 10 mM, causes 10-20% inhibition of carboxynorspermidine formation Vibrio alginolyticus
1.4.1.B4 cadaverine 10 mM, causes 10-20% inhibition of carboxynorspermidine formation Vibrio alginolyticus
1.4.1.B4 ethylenediamine 10 mM, causes 10-20% inhibition of carboxynorspermidine formation Vibrio alginolyticus
1.4.1.B4 ethylmaleimide 5 mM, in presence of 20 mM, 83% inhibition Vibrio alginolyticus
1.4.1.B4 iodoacetamide 5 mM, in presence of 20 mM, 24% inhibition Vibrio alginolyticus
1.4.1.B4 additional information no inhibitory effect of NAD+, ATP, and spermidine at 5 or 10 mM. No effect of 1 mM Na+ or K+. No evidence for inhibitory effect of metal cations such as Ca2+, Mg2+, Fe3+, Fe2+, Cu+, Cu2+, Mn2+, and Zn2+. EDTA, 1 mM no inhibition Vibrio alginolyticus
1.4.1.B4 NADP+ 5 mM, 51% inhibition Vibrio alginolyticus
1.4.1.B4 norspermidine 10 mM, 10% inhibition Vibrio alginolyticus
1.5.1.43 iodoacetamide 24% inhibition at 5 mM Vibrio alginolyticus
1.5.1.43 additional information EDTA (1 mM) shows no significant inhibitory effect on enzyme activity; NAD+, ATP and SPD are not inhibitory at 5 mM Vibrio alginolyticus
1.5.1.43 N-ethylmaleimide 83% inhibition at 5 mM Vibrio alginolyticus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.4.1.B4 additional information
-
 additional information the Km for the Schiff base is 4.68 mM, the intermediate formed from L-aspartic 4-semialdehyde and 1,3-diaminopropane, which is reduced to carboxynorspermidine Vibrio alginolyticus
1.4.1.B4 1.51
-
 NADPH pH 7.5, 37°C, with a Vmax of 31 micromol carboxynorspermidine/min/mg protein formed Vibrio alginolyticus
1.4.1.B4 2.97
-
 NADH pH 7.5, 37°C, with a Vmax of 13.5 micromol carboxynorspermidine/min/mg protein formed Vibrio alginolyticus
1.4.1.B4 4.68
-
 L-aspartic 4-semialdehyde pH 7.5, 37°C, Vmax: 35 micromol/min/mg carboxynorspermidine Vibrio alginolyticus
1.5.1.43 1.51
-
 NADPH at pH 7.5 and 37°C Vibrio alginolyticus
1.5.1.43 2.97
-
 NADH at pH 7.5 and 37°C Vibrio alginolyticus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.4.1.B4 additional information no evidence for activity being dependent on metal ions such as Ca2+, Mg2+, Fe3+, Fe2+, Cu+, Cu2+, Mn2+, and Zn2+ Vibrio alginolyticus
1.5.1.43 additional information Ca2+, Mg2+, Fe3+, Fe2+, Cu+, Cu2+, Mn2+, Zn2+, Na+ and K+ at 1 M have no effect on enzyme activity Vibrio alginolyticus

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.4.1.B4 45100
-
 2 * 45100, SDS-PAGE, indicates that the native enzyme is composed of two subunits of similar size Vibrio alginolyticus
1.4.1.B4 93500
-
 gel filtration Vibrio alginolyticus
1.5.1.43 45100
-
 2 * 45100, SDS-PAGE Vibrio alginolyticus
1.5.1.43 93500
-
 gel filtration Vibrio alginolyticus

Organism

EC Number Organism UniProt Comment Textmining
1.4.1.B4 Vibrio alginolyticus
-
-
-
1.5.1.43 Vibrio alginolyticus
-
-
-
1.5.1.43 Vibrio alginolyticus ATCC 17749
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.4.1.B4 to homogeneity, yield 33%, 1800fold purified, ammonium sulfate precipitation, DEAE-Sepharose CL-6B, hydroxyapatite, and Blue Sepharose CL-6B chromatography Vibrio alginolyticus
1.5.1.43 ammonium sulfate precipitation, Blue Sepharose CL-6B column chromatography, DEAE-Sepharose column chromatography, and hydroxyapatite column chromatography Vibrio alginolyticus

Reaction

EC Number Reaction Comment Organism Reaction ID
1.4.1.B4 L-aspartic 4-semialdehyde + 1,3-diaminopropane + NADPH + H+ = carboxynorspermidine + NADP+ + H2O i.e. (3-((3-aminopropyl)amino)propyl)carbamate (carboxynorspermidine) and (2S)-2-amino-4-oxobutanoate (L-aspartic 4-semialdehyde) via a nicotinamide-nucleotide reduction of the Schiff base H2N(CH2)3=CHCH2CH(NH2)COOH, formed from L-aspartic beta-semialdehyde, an intermediate in the novel pathway for norspermidine biosynthesis Vibrio alginolyticus
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.4.1.B4 additional information
-
 in the presence of 5 mM nospermidine, specific activity is reduced by 70% Vibrio alginolyticus
1.4.1.B4 0.0039
-
 addition of 5 mM norspermidine to the growth medium, 27% compared to the specific activity without addition, pH 7.5, 37°C Vibrio alginolyticus
1.4.1.B4 0.0108
-
 addition of 5 mM spermidine to the growth medium, 74% compared to the specific activity without addition, pH 7.5, 37°C Vibrio alginolyticus
1.4.1.B4 0.01455
-
 without addition of norspermidine or spermidine to the growth medium, pH 7.5, 37°C Vibrio alginolyticus
1.4.1.B4 31
-
 micromol carboxynorspermidine/min-1/mg protein, pH 7.5, 37°C Vibrio alginolyticus
1.5.1.43 0.0167
-
 crude enzyme, at pH 7.5 and 37°C Vibrio alginolyticus
1.5.1.43 31
-
 after 1856fold purification, at pH 7.5 and 37°C Vibrio alginolyticus

Storage Stability

EC Number Storage Stability Organism
1.4.1.B4 4°C, protein concentration of 1.6 mg/ml in 20 mM Tris-HCl, pH 7.5, 1 mM DTT and 0.02% NaN3, lost of about 15% of its activity after one week Vibrio alginolyticus
1.5.1.43 4°C, in 20 mM Tris/HCl, pH 7.5, containing 1 mM dithiothreitol and 0.02% (v/v) NaN3, 1 week, 15% loss of activity Vibrio alginolyticus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.4.1.B4 carboxynorspermidine + NAD+ + H2O NADH is a much poorer cofactor than NADPH Vibrio alginolyticus L-aspartic 4-semialdehyde + 1,3-diaminopropane + NADH + H+
-
 ir
1.4.1.B4 carboxynorspermidine + NAD+ + H2O NADH is a much poorer cofactor than NADPH Vibrio alginolyticus ATCC 17749 L-aspartic 4-semialdehyde + 1,3-diaminopropane + NADH + H+
-
 ir
1.4.1.B4 carboxynorspermidine + NADP+ + H2O i.e. (3-((3-aminopropyl)amino)propyl)carbamate, incubation of the purified enzyme in the range of pH 6-8, with cofactor NADP+ or NAD+ concentrations up to 5 mM and substrate concentrations of carboxynorspermidine up to 10 mM, no reversible activity is observed Vibrio alginolyticus L-aspartic 4-semialdehyde + 1,3-diaminopropane + NADPH + H+ i.e. (2S)-2-amino-4-oxobutanoate ir
1.4.1.B4 carboxynorspermidine + NADP+ + H2O i.e. (3-((3-aminopropyl)amino)propyl)carbamate, incubation of the purified enzyme in the range of pH 6-8, with cofactor NADP+ or NAD+ concentrations up to 5 mM and substrate concentrations of carboxynorspermidine up to 10 mM, no reversible activity is observed Vibrio alginolyticus ATCC 17749 L-aspartic 4-semialdehyde + 1,3-diaminopropane + NADPH + H+ i.e. (2S)-2-amino-4-oxobutanoate ir
1.4.1.B4 carboxyspermidine + NADP+ + H2O i.e. 3-[(4-aminobutyl)amino]-L-alanine, slightly active with putrescine, ca. 7% of the rate compared to 1,3-diaminopropane Vibrio alginolyticus L-aspartic 4-semialdehyde + putrescine + NADPH + H+
-
 ir
1.4.1.B4 carboxyspermidine + NADP+ + H2O i.e. 3-[(4-aminobutyl)amino]-L-alanine, slightly active with putrescine, ca. 7% of the rate compared to 1,3-diaminopropane Vibrio alginolyticus ATCC 17749 L-aspartic 4-semialdehyde + putrescine + NADPH + H+
-
 ir
1.4.1.B4 additional information no activity with ethylenediamine, cadaverine, and D-aspartic beta-semialdehyde Vibrio alginolyticus ?
-
 ?
1.4.1.B4 additional information no activity with ethylenediamine, cadaverine, and D-aspartic beta-semialdehyde Vibrio alginolyticus ATCC 17749 ?
-
 ?
1.5.1.43 L-aspartate 4-semialdehyde + propane-1,3-diamine + NADH + H+ NADH is a much poorer cofactor than NADPH Vibrio alginolyticus carboxynorspermidine + H2O + NAD+
-
 ?
1.5.1.43 L-aspartate 4-semialdehyde + propane-1,3-diamine + NADPH + H+
-
 Vibrio alginolyticus carboxynorspermidine + H2O + NADP+
-
 ?
1.5.1.43 L-aspartate 4-semialdehyde + putrescine + NADPH + H+ 7% activity with putrescine compared to propane-1,3-diamine Vibrio alginolyticus carboxyspermidine + H2O + NADP+
-
 ?
1.5.1.43 additional information does not react with ethylenediamine, cadaverine and D-aspartate 4-semialdehyde Vibrio alginolyticus ?
-
 ?
1.5.1.43 additional information does not react with ethylenediamine, cadaverine and D-aspartate 4-semialdehyde Vibrio alginolyticus ATCC 17749 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.4.1.B4 dimer 2 * 45100, SDS-PAGE, indicates that the native enzyme is composed of two subunits of similar size Vibrio alginolyticus
1.5.1.43 homodimer 2 * 45100, SDS-PAGE Vibrio alginolyticus

Synonyms

EC Number Synonyms Comment Organism
1.4.1.B4 C-NSPD synthase
-
 Vibrio alginolyticus
1.4.1.B4 carboxynorspermidine synthase
-
 Vibrio alginolyticus
1.5.1.43 ASA-DAP Schiff base reductase
-
 Vibrio alginolyticus
1.5.1.43 C-NSPD synthase
-
 Vibrio alginolyticus
1.5.1.43 carboxynorspermidine synthase
-
 Vibrio alginolyticus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.4.1.B4 37
-
-
 Vibrio alginolyticus
1.5.1.43 37
-
-
 Vibrio alginolyticus

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.4.1.B4 30 45 78 and 59% of the maximal activity at 30 and 45°C, respectively, no activity at 50°C Vibrio alginolyticus
1.5.1.43 30 50 78% and 59% of the maximum activity remain at 30°C and 45°C, respectively, but no activity is observed above 50°C Vibrio alginolyticus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.4.1.B4 7.25 7.5
-
 Vibrio alginolyticus
1.5.1.43 7.3 7.5
-
 Vibrio alginolyticus

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.4.1.B4 6.25 8 18 and 85% of the maximal acticity at pH 6.25 and 8.0, respectively Vibrio alginolyticus
1.5.1.43 6.3 8 85% and 18% of the maximum activity are observed at pH 8.0 and 6.3, respectively Vibrio alginolyticus

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.4.1.B4 6.5
-
 the enzyme is unstable in buffers below pH 6.5, at pH 6.5 50% loss of activity is observed within 12 h at 4°C Vibrio alginolyticus
1.5.1.43 6.5
-
 the enzyme is unstable in buffers of pH below 6.5. At pH 6.5, 50% loss of activity is observed within 12 h at 4°C Vibrio alginolyticus

Cofactor

EC Number Cofactor Comment Organism Structure
1.4.1.B4 NADH conmpletely inactive unless NADPH or NADH are added, NADH is a much poorer cofactor than NADPH Vibrio alginolyticus
1.4.1.B4 NADPH conmpletely inactive unless NADPH or NADH are added, NADH is a much poorer cofactor than NADPH Vibrio alginolyticus
1.5.1.43 NADH NADH is a much poorer cofactor than NADPH Vibrio alginolyticus
1.5.1.43 NADPH
-
 Vibrio alginolyticus

pI Value

EC Number Organism Comment pI Value Maximum pI Value
1.4.1.B4 Vibrio alginolyticus isoelectric focusing and chromatofocusing 4.3 4.2
1.5.1.43 Vibrio alginolyticus isoelectric focusing 4.3 4.2

Expression

EC Number Organism Comment Expression
1.5.1.43 Vibrio alginolyticus when Vibrio alginolyticus is grown in the presence of 5 mM norspermidine, the specific activity of the enzyme is reduced by about 70% down

General Information

EC Number General Information Comment Organism
1.4.1.B4 physiological function carboxynorspermidine synthase participates in the novel pathway for norspermidine biosynthesis, with two other enzymes, 2,4-diamonobutyrate deacroxylase and carboxynorspermidine decarboxylase Vibrio alginolyticus