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Literature summary for 3.5.1.19 extracted from

  • Sanchez-Carron, G.; Garcia-Garcia, M.I.; Zapata-Perez, R.; Takami, H.; Garcia-Carmona, F.; Sanchez-Ferrer, A.
    Biochemical and mutational analysis of a novel nicotinamidase from Oceanobacillus iheyensis HTE831 (2013), PLoS ONE, 8, e56727.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta 2 (DE3) Oceanobacillus iheyensis

Protein Variants

Protein Variants Comment Organism
C133A site-directed mutagenesis, the mutation affectes the active site cavity, and enhances the binding of pyrazinamide compared to the wild-type enzyme Oceanobacillus iheyensis
E65H site-directed mutagenesis, the mutation highly reduces the activity with pyrazinamide compared to the wild-type enzyme Oceanobacillus iheyensis
F68W site-directed mutagenesis, the mutation affectes the active site cavity, and enhances the binding of pyrazinamide compared to the wild-type enzyme Oceanobacillus iheyensis
F68W/C133A site-directed mutagenesis, the mutation affectes the active site cavity, and enhances the binding of pyrazinamide compared to the wild-type enzyme Oceanobacillus iheyensis
K104A site-directed mutagenesis, the mutant shows 98.55% reduced activity compared to the wild-type enzyme Oceanobacillus iheyensis
additional information mutation of residues Cys133 and Phe68 is required for increasing pyrazinamidase activity 2.9 and 2.5fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) is detrimental to pyrazinamidase activity decreasing it 6fold Oceanobacillus iheyensis
Q96A site-directed mutagenesis, the mutant shows similar activity with nicotinamide and increased activity with 5-methyl nicotinamide compared to the wild-type enzyme Oceanobacillus iheyensis
Q96K site-directed mutagenesis, the mutant shows 62.7% reduced activity compared to the wild-type enzyme Oceanobacillus iheyensis
T12Q site-directed mutagenesis, the mutant shows 98.42% reduced activity compared to the wild-type enzyme Oceanobacillus iheyensis

Inhibitors

Inhibitors Comment Organism Structure
5-bromonicotinaldehyde competitive inhibition Oceanobacillus iheyensis
nicotinaldehyde competitive inhibition Oceanobacillus iheyensis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Oceanobacillus iheyensis
0.13
-
nicotinamide recombinant mutant E65H, pH 7.3, 37°C Oceanobacillus iheyensis
0.17
-
nicotinamide recombinant mutant F68W, pH 7.3, 37°C Oceanobacillus iheyensis
0.19
-
nicotinamide recombinant mutant F68W/C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.26
-
nicotinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.34
-
ethyl nicotinate recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.36
-
Pyrazinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.36
-
nicotinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
0.4
-
nicotinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.44
-
methyl nicotinate recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.5
-
Pyrazinamide recombinant mutant F68W, pH 7.3, 37°C Oceanobacillus iheyensis
0.55
-
5-methyl nicotinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
0.56
-
methyl nicotinate recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
0.57
-
nicotinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.62
-
5-methyl nicotinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.64
-
5-methyl nicotinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.68
-
5-methyl nicotinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.81
-
Pyrazinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.82
-
Pyrazinamide recombinant mutant E65H, pH 7.3, 37°C Oceanobacillus iheyensis
0.83
-
Pyrazinamide recombinant mutant F68W/C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.89
-
methyl nicotinate recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
1.02
-
ethyl nicotinate recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
1.03
-
methyl nicotinate recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
1.11
-
ethyl nicotinate recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
1.15
-
Pyrazinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
1.2
-
Pyrazinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
1.43
-
ethyl nicotinate recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis

Metals/Ions

Metals/Ions Comment Organism Structure
additional information the enzyme contains 0.235 mM of Zn2+ in 0.242 mM protein, while it only contains 0.0032 mM of Fe2+ and 0.00077 mM Mn2+ Oceanobacillus iheyensis
Zn2+ presence of Zn2+ in the active center of the enzyme in a molecular ratio of 1:1 of protein to metal ion Oceanobacillus iheyensis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
21100
-
2 * 21100, mass spectrometry Oceanobacillus iheyensis
42900
-
gel filtration Oceanobacillus iheyensis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
nicotinamide + H2O Oceanobacillus iheyensis
-
nicotinate + NH3
-
?
nicotinamide + H2O Oceanobacillus iheyensis HTE831
-
nicotinate + NH3
-
?
pyrazinamide + H2O Oceanobacillus iheyensis
-
pyrazinoic acid + NH3
-
?
pyrazinamide + H2O Oceanobacillus iheyensis HTE831
-
pyrazinoic acid + NH3
-
?

Organism

Organism UniProt Comment Textmining
Oceanobacillus iheyensis Q8ESQ6 deep-sea extremely halotolerant and alkaliphilic organism
-
Oceanobacillus iheyensis HTE831 Q8ESQ6 deep-sea extremely halotolerant and alkaliphilic organism
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes methylnicotinamide from Escherichia coli strain Rosetta 2 (DE3) by ultrafiltration and nickel affinity chromatography Oceanobacillus iheyensis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.06
-
purified recombinant mutant Q96K, pH 7.3, 37°C, substrate ethyl nicotinate Oceanobacillus iheyensis
0.13
-
purified recombinant wild-type enzyme, pH 7.3, 37°C, substrate ethyl nicotinate Oceanobacillus iheyensis
0.22
-
purified recombinant mutant Q96A, pH 7.3, 37°C, substrate ethyl nicotinate Oceanobacillus iheyensis
0.31
-
purified recombinant mutant Q96K, pH 7.3, 37°C, substrate methyl nicotinate Oceanobacillus iheyensis
0.34
-
purified recombinant mutant C133A, pH 7.3, 37°C, substrate ethyl nicotinate Oceanobacillus iheyensis
0.36
-
purified recombinant mutant K104A, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
0.37
-
purified recombinant mutant T12Q, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
0.45
-
purified recombinant wild-type enzyme, pH 7.3, 37°C, substrate methyl nicotinate Oceanobacillus iheyensis
0.46
-
purified recombinant mutant Q96K, pH 7.3, 37°C, substrate pyrazinamide Oceanobacillus iheyensis
0.7
-
purified recombinant mutant E65H, pH 7.3, 37°C, substrate pyrazinamide Oceanobacillus iheyensis
1.16
-
purified recombinant mutant Q96A, pH 7.3, 37°C, substrate methyl nicotinate Oceanobacillus iheyensis
1.4
-
purified recombinant mutant C133A, pH 7.3, 37°C, substrate methyl nicotinate Oceanobacillus iheyensis
1.8
-
purified recombinant mutant Q96A, pH 7.3, 37°C, substrate pyrazinamide Oceanobacillus iheyensis
3.62
-
purified recombinant wild-type enzyme, pH 7.3, 37°C, substrate pyrazinamide Oceanobacillus iheyensis
3.96
-
purified recombinant mutant Q96K, pH 7.3, 37°C, substrate 5-methyl nicotinamide Oceanobacillus iheyensis
6.39
-
purified recombinant mutant C133A, pH 7.3, 37°C, substrate pyrazinamide Oceanobacillus iheyensis
7.43
-
purified recombinant mutant F68W/C133A, pH 7.3, 37°C, substrate pyrazinamide Oceanobacillus iheyensis
7.5
-
purified recombinant mutant F68W, pH 7.3, 37°C, substrate pyrazinamide Oceanobacillus iheyensis
8.7
-
purified recombinant mutant Q96K, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
9.01
-
purified recombinant wild-type enzyme, pH 7.3, 37°C, substrate 5-methyl nicotinamide Oceanobacillus iheyensis
11.74
-
purified recombinant mutant C133A, pH 7.3, 37°C, substrate 5-methyl nicotinamide Oceanobacillus iheyensis
13.5
-
purified recombinant mutant E65H, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
17.5
-
purified recombinant mutant C133A, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
19.97
-
purified recombinant mutant F68W, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
22.9
-
purified recombinant mutant F68W/C133A, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
23.3 25 purified recombinant wild-type enzyme, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
24.8
-
purified recombinant mutant Q96A, pH 7.3, 37°C, substrate nicotinamide Oceanobacillus iheyensis
28
-
purified recombinant mutant Q96A, pH 7.3, 37°C, substrate 5-methyl nicotinamide Oceanobacillus iheyensis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5-methyl nicotinamide + H2O
-
Oceanobacillus iheyensis 5-methyl nicotinate + NH3
-
?
5-methyl nicotinamide + H2O
-
Oceanobacillus iheyensis HTE831 5-methyl nicotinate + NH3
-
?
ethyl nicotinate + H2O
-
Oceanobacillus iheyensis nicotinate + ethanol
-
?
methyl nicotinate + H2O
-
Oceanobacillus iheyensis nicotinate + methanol
-
?
nicotinamide + H2O
-
Oceanobacillus iheyensis nicotinate + NH3
-
?
nicotinamide + H2O preferred substrate Oceanobacillus iheyensis nicotinate + NH3
-
?
nicotinamide + H2O
-
Oceanobacillus iheyensis HTE831 nicotinate + NH3
-
?
nicotinamide + H2O preferred substrate Oceanobacillus iheyensis HTE831 nicotinate + NH3
-
?
pyrazinamide + H2O
-
Oceanobacillus iheyensis pyrazinoic acid + NH3
-
?
pyrazinamide + H2O residues Cys133 and Phe68 are critical for the pyraminidase activity Oceanobacillus iheyensis pyrazinoic acid + NH3
-
?
pyrazinamide + H2O
-
Oceanobacillus iheyensis HTE831 pyrazinoic acid + NH3
-
?
pyrazinamide + H2O residues Cys133 and Phe68 are critical for the pyraminidase activity Oceanobacillus iheyensis HTE831 pyrazinoic acid + NH3
-
?

Subunits

Subunits Comment Organism
dimer 2 * 21100, mass spectrometry Oceanobacillus iheyensis

Synonyms

Synonyms Comment Organism
OiNIC
-
Oceanobacillus iheyensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
45
-
-
Oceanobacillus iheyensis

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
25 55 activity range, below 25°C or above 55°C the enzyme loses most of its activity Oceanobacillus iheyensis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
4 20 purified recombinant wild-type enzyme, 20 h, stable Oceanobacillus iheyensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.05
-
ethyl nicotinate recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.1
-
ethyl nicotinate recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.14
-
ethyl nicotinate recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.17
-
methyl nicotinate recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.19
-
ethyl nicotinate recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
0.32
-
methyl nicotinate recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.43
-
Pyrazinamide recombinant mutant E65H, pH 7.3, 37°C Oceanobacillus iheyensis
0.45
-
Pyrazinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.6
-
methyl nicotinate recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
1.02
-
methyl nicotinate recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
1.61
-
Pyrazinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
2.16
-
5-methyl nicotinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
2.37
-
nicotinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
2.6
-
Pyrazinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
3.31
-
Pyrazinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
3.94
-
Pyrazinamide recombinant mutant F68W, pH 7.3, 37°C Oceanobacillus iheyensis
5.1
-
5-methyl nicotinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
5.21
-
Pyrazinamide recombinant mutant F68W/C133A, pH 7.3, 37°C Oceanobacillus iheyensis
5.63
-
nicotinamide recombinant mutant E65H, pH 7.3, 37°C Oceanobacillus iheyensis
7.09
-
5-methyl nicotinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
8.79
-
nicotinamide recombinant mutant F68W, pH 7.3, 37°C Oceanobacillus iheyensis
9.82
-
nicotinamide recombinant mutant F68W/C133A, pH 7.3, 37°C Oceanobacillus iheyensis
11.65
-
nicotinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
13.6
-
nicotinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
15.9
-
5-methyl nicotinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
16.05
-
nicotinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6 6.5
-
Oceanobacillus iheyensis

pH Range

pH Minimum pH Maximum Comment Organism
5 8 activity range, marked decrease in activity below pH 5.0 and above pH 8.0 Oceanobacillus iheyensis

pH Stability

pH Stability pH Stability Maximum Comment Organism
6 7.3 purified recombinant wild-type enzyme, very stable at pH 6.0 and pH 7.3, where it maintains 40% and 30% residual activity, respectively, after 20 hours of incubation Oceanobacillus iheyensis

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.0035
-
nicotinaldehyde recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.0044
-
5-bromonicotinaldehyde recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis

General Information

General Information Comment Organism
metabolism nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD+ salvage pathway Oceanobacillus iheyensis
additional information two for residues involved in the catalysis (K104 and Q96), one for the fourth residue involved in metal binding (E65), one for the residue forming hydrogen bonds between the main and lateral chains (T12), one for the residue involved in the cis-peptide bond and in the formation of the oxyanion hole (C133), and one for the residue forming one of the faces of the active site F68 Oceanobacillus iheyensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.05
-
ethyl nicotinate recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.1
-
ethyl nicotinate recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.13
-
ethyl nicotinate recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
0.31
-
methyl nicotinate recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
0.38
-
methyl nicotinate recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.39
-
Pyrazinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
0.41
-
ethyl nicotinate recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
0.52
-
Pyrazinamide recombinant mutant E65H, pH 7.3, 37°C Oceanobacillus iheyensis
1.14
-
methyl nicotinate recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
1.17
-
methyl nicotinate recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
1.34
-
Pyrazinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
3.2
-
Pyrazinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
3.4
-
5-methyl nicotinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
4.15
-
nicotinamide recombinant mutant Q96K, pH 7.3, 37°C Oceanobacillus iheyensis
6.28
-
Pyrazinamide recombinant mutant F68W/C133A, pH 7.3, 37°C Oceanobacillus iheyensis
7.5
-
5-methyl nicotinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
7.9
-
Pyrazinamide recombinant mutant F68W, pH 7.3, 37°C Oceanobacillus iheyensis
9.19
-
Pyrazinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
11.07
-
5-methyl nicotinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
29.04
-
5-methyl nicotinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
34
-
nicotinamide recombinant mutant C133A, pH 7.3, 37°C Oceanobacillus iheyensis
42.65
-
nicotinamide recombinant mutant E65H, pH 7.3, 37°C Oceanobacillus iheyensis
43.48
-
nicotinamide recombinant wild-type enzyme, pH 7.3, 37°C Oceanobacillus iheyensis
44.59
-
nicotinamide recombinant mutant Q96A, pH 7.3, 37°C Oceanobacillus iheyensis
52.01
-
nicotinamide recombinant mutant F68W, pH 7.3, 37°C Oceanobacillus iheyensis
52.8
-
nicotinamide recombinant mutant F68W/C133A, pH 7.3, 37°C Oceanobacillus iheyensis