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Literature summary for 1.4.1.B4 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

Activating Compound Comment Organism Structure
DTT without addition 7% of the maximum activity detected in the purified enzyme, 14fold activiation is observed at 20 mM Vibrio alginolyticus
additional information 1 mM EDTA shows no stimulatory effect on enzyme activity Vibrio alginolyticus

General Stability

General Stability Organism
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

Inhibitors Comment Organism Structure
1,2-diaminopropane 10 mM, causes 10-20% inhibition of carboxynorspermidine formation Vibrio alginolyticus
cadaverine 10 mM, causes 10-20% inhibition of carboxynorspermidine formation Vibrio alginolyticus
ethylenediamine 10 mM, causes 10-20% inhibition of carboxynorspermidine formation Vibrio alginolyticus
ethylmaleimide 5 mM, in presence of 20 mM, 83% inhibition Vibrio alginolyticus
iodoacetamide 5 mM, in presence of 20 mM, 24% inhibition Vibrio alginolyticus
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
NADP+ 5 mM, 51% inhibition Vibrio alginolyticus
norspermidine 10 mM, 10% inhibition Vibrio alginolyticus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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
1.51
-
NADPH pH 7.5, 37°C, with a Vmax of 31 micromol carboxynorspermidine/min/mg protein formed Vibrio alginolyticus
2.97
-
NADH pH 7.5, 37°C, with a Vmax of 13.5 micromol carboxynorspermidine/min/mg protein formed Vibrio alginolyticus
4.68
-
L-aspartic 4-semialdehyde pH 7.5, 37°C, Vmax: 35 micromol/min/mg carboxynorspermidine Vibrio alginolyticus

Metals/Ions

Metals/Ions Comment Organism Structure
additional information no evidence for activity being dependent on metal ions such as Ca2+, Mg2+, Fe3+, Fe2+, Cu+, Cu2+, Mn2+, and Zn2+ Vibrio alginolyticus

Molecular Weight [Da]

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

Organism

Organism UniProt Comment Textmining
Vibrio alginolyticus
-
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
in the presence of 5 mM nospermidine, specific activity is reduced by 70% Vibrio alginolyticus
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
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
0.01455
-
without addition of norspermidine or spermidine to the growth medium, pH 7.5, 37°C Vibrio alginolyticus
31
-
micromol carboxynorspermidine/min-1/mg protein, pH 7.5, 37°C Vibrio alginolyticus

Storage Stability

Storage Stability Organism
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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
carboxynorspermidine + NAD+ + H2O NADH is a much poorer cofactor than NADPH Vibrio alginolyticus L-aspartic 4-semialdehyde + 1,3-diaminopropane + NADH + H+
-
ir
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
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
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
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
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
additional information no activity with ethylenediamine, cadaverine, and D-aspartic beta-semialdehyde Vibrio alginolyticus ?
-
?
additional information no activity with ethylenediamine, cadaverine, and D-aspartic beta-semialdehyde Vibrio alginolyticus ATCC 17749 ?
-
?

Subunits

Subunits Comment Organism
dimer 2 * 45100, SDS-PAGE, indicates that the native enzyme is composed of two subunits of similar size Vibrio alginolyticus

Synonyms

Synonyms Comment Organism
C-NSPD synthase
-
Vibrio alginolyticus
carboxynorspermidine synthase
-
Vibrio alginolyticus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
-
Vibrio alginolyticus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
30 45 78 and 59% of the maximal activity at 30 and 45°C, respectively, no activity at 50°C Vibrio alginolyticus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.25 7.5
-
Vibrio alginolyticus

pH Range

pH Minimum pH Maximum Comment Organism
6.25 8 18 and 85% of the maximal acticity at pH 6.25 and 8.0, respectively Vibrio alginolyticus

pH Stability

pH Stability pH Stability Maximum Comment Organism
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

Cofactor

Cofactor Comment Organism Structure
NADH conmpletely inactive unless NADPH or NADH are added, NADH is a much poorer cofactor than NADPH Vibrio alginolyticus
NADPH conmpletely inactive unless NADPH or NADH are added, NADH is a much poorer cofactor than NADPH Vibrio alginolyticus

pI Value

Organism Comment pI Value Maximum pI Value
Vibrio alginolyticus isoelectric focusing and chromatofocusing 4.3 4.2

General Information

General Information Comment Organism
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