Literature summary for 3.5.1.19 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 .

Application

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

Cloned(Commentary)

Cloned (Comment)	Organism
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
expression in Escherichia coli Rosetta 2DE3	unidentified

Cloned (Comment)

Organism

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

expression in Escherichia coli Rosetta 2DE3

unidentified

Inhibitors

Inhibitors	Comment	Organism
5-bromo-nicotinaldehyde	-	uncultured bacterium
5-bromonicotinaldehyde	-	unidentified
nicotinaldehyde	-	uncultured bacterium
nicotinaldehyde	-	unidentified

KM Value [mM]

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

Metals/Ions

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

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

Purification (Comment)	Organism
-	unidentified

Substrates and Products (Substrate)

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

Subunits

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

Synonyms

Synonyms	Comment	Organism
polygenomic nicotinamidase	-	unidentified
PolyNic	-	unidentified
PolyNic	-	uncultured bacterium

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
50	-	-	unidentified
50	-	at 37°C	uncultured bacterium

Temperature Range [°C]

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

Turnover Number [1/s]

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

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
10	-	-	unidentified
10	-	at pH 8.0	uncultured bacterium

pH Range

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

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.000015	-	nicotinaldehyde	pH 7.3, temperature not specified in the publication, substrate: pyrazinamide	unidentified
0.00007	-	5-bromonicotinaldehyde	pH 7.3, temperature not specified in the publication, substrate: pyrazinamide	unidentified

General Information

General Information	Comment	Organism
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
physiological function	the enzyme is an essential component of the NAD+ salvage pathway	unidentified
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]

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