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 | 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 | Comment | Organism | Structure |
---|---|---|---|
5-bromonicotinaldehyde | competitive inhibition | Oceanobacillus iheyensis | |
nicotinaldehyde | competitive inhibition | Oceanobacillus iheyensis |
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 | 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 Maximum [Da] | Comment | Organism |
---|---|---|---|
21100 | - |
2 * 21100, mass spectrometry | Oceanobacillus iheyensis |
42900 | - |
gel filtration | Oceanobacillus iheyensis |
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 | 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 (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 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 | 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 | Comment | Organism |
---|---|---|
dimer | 2 * 21100, mass spectrometry | Oceanobacillus iheyensis |
Synonyms | Comment | Organism |
---|---|---|
OiNIC | - |
Oceanobacillus iheyensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
45 | - |
- |
Oceanobacillus iheyensis |
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 Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
4 | 20 | purified recombinant wild-type enzyme, 20 h, stable | Oceanobacillus iheyensis |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | 6.5 | - |
Oceanobacillus iheyensis |
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 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 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 | 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 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 |