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Literature summary extracted from

  • Zapata-Perez, R.; Garcia-Saura, A.G.; Jebbar, M.; Golyshin, P.N.; Sanchez-Ferrer, A.
    Combined whole-cell high-throughput functional screening for identification of new nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries (2016), Front. Microbiol., 7, 1915 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
3.5.1.19 analysis nicotinamidases are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases uncultured bacterium
3.5.1.19 medicine the absence of nicotinamidase in humans makes it a promising drug target unidentified

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.B15 combined whole-cell high-throughput functional screening for identification of nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries. A fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria is screened, discovering several positive clones with the ammonium ferrous sulfate method. Quantitative rescreening with the SNP method allowing the finding of the first nicotinamidase with balanced catalytic efficiency toward nicotinamidase activity (EC 3.5.1.19) and pyrazinamide (pyrazinamidase activity). Escherichia coli EPI300-T1 is used for the screening of the genomic libraries cloned in pCC1FOS fosmids library. Escherichia coli strain Rosetta2 (DE3) is used as the host for pET46Ek/LIC expression vector. The fosmid library contains inserts of mixed genomic DNA of 194 mesophilic bacteria (MB) isolated from deep-sea hydrothermal vents sampled at various sites in Mid Atlantic Ridge, genotyping, DNA and amino acid sequence determination and analysis uncultured bacterium
3.5.1.19 combined whole-cell high-throughput functional screening for identification of nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries. A fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria is screened, discovering several positive clones with the ammonium ferrous sulfate method. Quantitative rescreening with the SNP method allowing the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). Escherichia coli EPI300-T1 is used for the screening of the genomic libraries cloned in pCC1FOS fosmids library. Escherichia coli strain Rosetta2 (DE3) is used as the host for pET46Ek/LIC expression vector. The fosmid library contains inserts of mixed genomic DNA of 194 mesophilic bacteria (MB) isolated from deep-sea hydrothermal vents sampled at various sites in Mid Atlantic Ridge, genotyping, DNA and amino acid sequence determination and analysis uncultured bacterium
3.5.1.19 expression in Escherichia coli Rosetta 2DE3 unidentified

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.1.B15 5-bromo-nicotinaldehyde
-
uncultured bacterium
3.5.1.B15 nicotinaldehyde
-
uncultured bacterium
3.5.1.19 5-bromo-nicotinaldehyde
-
uncultured bacterium
3.5.1.19 5-bromonicotinaldehyde
-
unidentified
3.5.1.19 nicotinaldehyde
-
uncultured bacterium
3.5.1.19 nicotinaldehyde
-
unidentified

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.19 0.049
-
nicotinamide pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 0.103
-
Pyrazinamide pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 0.355
-
methylnicotinate pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 0.435
-
5-Methylnicotinamide pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 1.092
-
ethylnicotinate pH 7.3, temperature not specified in the publication unidentified

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.5.1.19 Fe2+ the conserved specific metal ion binding includes one aspartate (D67) and two histidines (H69 and H86) uncultured bacterium

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.5.1.19 50300
-
gel filtration unidentified

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.B15 pyrazinamide + H2O uncultured bacterium
-
pyrazinoic acid + NH3
-
r
3.5.1.19 nicotinamide + H2O unidentified the enzyme is an essential component of the NAD+ salvage pathway nicotinate + NH3
-
?
3.5.1.19 nicotinamide + H2O uncultured bacterium
-
nicotinic acid + NH3
-
r

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.B15 uncultured bacterium
-
mesophilic bacteria (MB) isolated from deep-sea hydrothermal vents sampled at various sites in Mid Atlantic Ridge
-
3.5.1.19 no activity in Homo sapiens
-
-
-
3.5.1.19 uncultured bacterium
-
mesophilic bacteria (MB) isolated from deep-sea hydrothermal vents sampled at various sites in Mid Atlantic Ridge
-
3.5.1.19 unidentified
-
marine metagenome
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.19
-
unidentified

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.B15 pyrazinamide + H2O
-
uncultured bacterium pyrazinoic acid + NH3
-
r
3.5.1.19 5-methylnicotinamide + H2O
-
unidentified 5-methylnicotinate + NH3
-
?
3.5.1.19 5-methylnicotinamide + H2O
-
uncultured bacterium 5-methylnicotinic acid + NH3
-
r
3.5.1.19 ethylnicotinate + H2O
-
unidentified nicotinate + ethanol
-
?
3.5.1.19 ethylnicotinate + H2O
-
uncultured bacterium nicotinic acid + ethanol
-
r
3.5.1.19 methylnicotinate + H2O
-
unidentified nicotinate + methanol
-
?
3.5.1.19 nicotinamide + H2O
-
unidentified nicotinate + NH3
-
?
3.5.1.19 nicotinamide + H2O the enzyme is an essential component of the NAD+ salvage pathway unidentified nicotinate + NH3
-
?
3.5.1.19 nicotinamide + H2O
-
uncultured bacterium nicotinic acid + NH3
-
r
3.5.1.19 pyrazinamide + H2O
-
unidentified pyrazinoic acid + NH3
-
?

Subunits

EC Number Subunits Comment Organism
3.5.1.19 dimer 2 * 23000, SDS-PAGE unidentified

Synonyms

EC Number Synonyms Comment Organism
3.5.1.B15 More cf. EC 3.5.1.19 uncultured bacterium
3.5.1.B15 PolyNic
-
uncultured bacterium
3.5.1.19 polygenomic nicotinamidase
-
unidentified
3.5.1.19 PolyNic
-
unidentified
3.5.1.19 PolyNic
-
uncultured bacterium

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.B15 37
-
assay at uncultured bacterium
3.5.1.19 50
-
-
unidentified
3.5.1.19 50
-
at 37°C uncultured bacterium

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3.5.1.19 10 90 activity range, profile overview uncultured bacterium
3.5.1.19 37 55 37°C: about 50% of maximal activity, 55°C: about 60% of maximal activity unidentified

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.19 1.1
-
ethylnicotinate pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 5
-
nicotinamide pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 7
-
methylnicotinate pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 7.2
-
Pyrazinamide pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 8.2
-
5-Methylnicotinamide pH 7.3, temperature not specified in the publication unidentified

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.B15 7.3
-
assay at uncultured bacterium
3.5.1.19 10
-
-
unidentified
3.5.1.19 10
-
at pH 8.0 uncultured bacterium

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.5.1.19 4 11 activity range, profile overview uncultured bacterium
3.5.1.19 6 10.5 pH 6.0: about 40% of maximal activity, pH 10.5: about 45% of maximal activity unidentified

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
3.5.1.B15 0.000015
-
nicotinaldehyde pH 7.3, 37°C uncultured bacterium
3.5.1.B15 0.00007
-
5-bromo-nicotinaldehyde pH 7.3, 37°C uncultured bacterium
3.5.1.19 0.000015
-
nicotinaldehyde pH 7.3, temperature not specified in the publication, substrate: pyrazinamide unidentified
3.5.1.19 0.00007
-
5-bromonicotinaldehyde pH 7.3, temperature not specified in the publication, substrate: pyrazinamide unidentified

General Information

EC Number General Information Comment Organism
3.5.1.B15 additional information combined whole-cell high-throughput functional screening for identification of nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries, screening of mesophilic marine bacteria (MB) polygenomic library. Development of two whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP), optimization of the assay. A fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria is screened, discovering several positive clones with the ammonium ferrous sulfate method. Quantitative rescreening with the SNP method allowing the finding of the first nicotinamidase with balanced catalytic efficiency toward nicotinamidase activity (EC 3.5.1.19) and pyrazinamide (pyrazinamidase activity) uncultured bacterium
3.5.1.19 additional information combined whole-cell high-throughput functional screening for identification of nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries, screening of mesophilic marine bacteria (MB) polygenomic library. Development of two whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP), optimization of the assay. A fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria is screened, discovering several positive clones with the ammonium ferrous sulfate method. Quantitative rescreening with the SNP method allowing the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). PolyNic contains all the conserved catalytic triad residues of the cysteine-hydrolases family:a catalytic cysteine at position 150, an aspartate at position 25, and a lysine at position 111. Other residues important for substrate and metal binding are F30, L36, W83, A112, Y120, and V149 uncultured bacterium
3.5.1.19 physiological function the enzyme is an essential component of the NAD+ salvage pathway unidentified
3.5.1.19 physiological function nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide (NAM) to produce ammonia and nicotinic acid (NA). These enzymes are an essential component of the NAD+ salvage pathway and are implicated in the viability of several pathogenic organisms uncultured bacterium

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.1.19 1
-
ethylnicotinate pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 18.9
-
5-Methylnicotinamide pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 19.7
-
methylnicotinate pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 69.9
-
Pyrazinamide pH 7.3, temperature not specified in the publication unidentified
3.5.1.19 102
-
nicotinamide pH 7.3, temperature not specified in the publication unidentified