Literature summary for 3.5.1.B15 extracted from

Rueda, D.; Bernard, C.; Gandy, L.; Capton, E.; Boudjelloul, R.; Brossier, F.; Veziris, N.; Zimic, M.; Sougakoff, W.
Estimation of pyrazinamidase activity using a cell-free in vitro synthesis of pnca and its association with pyrazinamide susceptibility in Mycobacterium tuberculosis (2018), Int. J. Mycobacteriol., 7, 16-25 .

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Cloned(Commentary)

Cloned (Comment)	Organism
gene pncA, PCR-based in vitro expression, and recombinant expression of His-tagged mutant enzyme in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain TOP10	Mycobacterium tuberculosis variant bovis
gene pncA, PCR-based in vitro expression, and recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain TOP10	Mycobacterium tuberculosis

Protein Variants

Protein Variants	Comment	Organism
A146V	naturally occuring mutation, the mutant shows 72% reduced activity compared to wild-type, resistant strain	Mycobacterium tuberculosis
D63A	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
G97D	naturally occuring mutation, the mutant shows 90% reduced activity compared to wild-type, resistant strain	Mycobacterium tuberculosis
H51P	naturally occuring mutation, inactive mutant, resistant strain	Mycobacterium tuberculosis
H51R	naturally occuring mutation, inactive mutant, resistant strain	Mycobacterium tuberculosis
H57D	site-directed mutagenesis, inactive mutant	Mycobacterium tuberculosis variant bovis
H57D	naturally occuring mutation, inactive mutant, resistant strain	Mycobacterium tuberculosis
I5T	naturally occuring mutation, the mutant shows 88% reduced activity compared to wild-type, resistant strain	Mycobacterium tuberculosis
I6L	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
L85R	naturally occuring mutation, inactive mutant, resistant strain	Mycobacterium tuberculosis
M175V	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
additional information	analysis of the specific pyrazinamidase (PZase) activity of PncA mutants via PCR-based in vitro-synthesized-PZase assay and correlation of the results to the pyrazinamide (PZA) susceptibility phenotype determined by culture in acidic agar medium at pH 6.0. A set of 23 clinical isolates displaying mutated pncA genes (11 PZA-resistant and 12 PZA-susceptible) and 55 PZA-susceptible clinical strains displaying a wild-type pncA gene are tested. Among the 23 mutants tested, 4 corresponded to mutations not reported before (I5T, Y99S, T142R, and P77L/V131G). Of the 11 PncA mutants expressed from PZA-resistant clinical isolates, 9 are expressed in vitro at yields above 50% relative to the wild-type enzyme. Among them, 6 enzymes (T47P, H51P, H51R, H57D, L85R and T142R) show no detectable activity, while the relative activities for the 3 others, V9A, G97D, and A146V, are low compared to the wild-type PZase. The remaining two mutants, I5T and V9G, presented very low relative expression (5%) and relative activities values of 12 and 1%, respectively. Twelve mutants are expressed from PZA-susceptible isolates. Their expression arte is similar to the wild-type enzyme, and they behave as active pyrazinamidase with specific relative activities ranging from 34 to 314%. Finally, discrepant results are observed for two mutants, V7A and P62T	Mycobacterium tuberculosis
P62T	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
P77L/V131G	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
R140S	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
T142R	naturally occuring mutation, inactive mutant, resistant strain	Mycobacterium tuberculosis
T142R	naturally occuring mutation, resistant strain	Mycobacterium tuberculosis
T167I	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
T47A	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
T47P	naturally occuring mutation, inactive mutant, resistant strain	Mycobacterium tuberculosis
T87M	site-directed mutagenesis, active mutant, susceptible strain	Mycobacterium tuberculosis
V7A	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
V9A	naturally occuring mutation, the mutant shows 73% reduced activity compared to wild-type, resistant strain	Mycobacterium tuberculosis
V9G	naturally occuring mutation, the mutant shows 99% reduced activity compared to wild-type, resistant strain	Mycobacterium tuberculosis
Y64D	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis
Y99S	naturally occuring mutation, susceptible strain	Mycobacterium tuberculosis

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	required, enzyme-bound	Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
pyrazinamide + H2O	Mycobacterium tuberculosis variant bovis	-	pyrazinoic acid + NH3	-	?
pyrazinamide + H2O	Mycobacterium tuberculosis	-	pyrazinoic acid + NH3	-	?
pyrazinamide + H2O	Mycobacterium tuberculosis H37Rv	-	pyrazinoic acid + NH3	-	?
pyrazinamide + H2O	Mycobacterium tuberculosis ATCC 25618	-	pyrazinoic acid + NH3	-	?

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	I6XD65	-	-
Mycobacterium tuberculosis ATCC 25618	I6XD65	-	-
Mycobacterium tuberculosis H37Rv	I6XD65	-	-
Mycobacterium tuberculosis variant bovis	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged mutant enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration	Mycobacterium tuberculosis variant bovis
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration	Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	colorimetric assay using ferrous ammonium sulfate 20% and glycine-HCl	Mycobacterium tuberculosis variant bovis	?	-	-
additional information	colorimetric assay using ferrous ammonium sulfate 20% and glycine-HCl	Mycobacterium tuberculosis	?	-	-
additional information	colorimetric assay using ferrous ammonium sulfate 20% and glycine-HCl	Mycobacterium tuberculosis H37Rv	?	-	-
additional information	colorimetric assay using ferrous ammonium sulfate 20% and glycine-HCl	Mycobacterium tuberculosis ATCC 25618	?	-	-
pyrazinamide + H2O	-	Mycobacterium tuberculosis variant bovis	pyrazinoic acid + NH3	-	?
pyrazinamide + H2O	-	Mycobacterium tuberculosis	pyrazinoic acid + NH3	-	?
pyrazinamide + H2O	-	Mycobacterium tuberculosis H37Rv	pyrazinoic acid + NH3	-	?
pyrazinamide + H2O	-	Mycobacterium tuberculosis ATCC 25618	pyrazinoic acid + NH3	-	?

Synonyms

Synonyms	Comment	Organism
More	cf. EC 3.5.1.19	Mycobacterium tuberculosis variant bovis
More	cf. EC 3.5.1.19	Mycobacterium tuberculosis
PncA	-	Mycobacterium tuberculosis variant bovis
PncA	-	Mycobacterium tuberculosis
pyrazinamidase/nicotinamidase	-	Mycobacterium tuberculosis variant bovis
pyrazinamidase/nicotinamidase	-	Mycobacterium tuberculosis
PZAse	-	Mycobacterium tuberculosis variant bovis
PZAse	-	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
malfunction	estimation of pyrazinamidase activity using a cell-free in vitro synthesis of pncA and its association with pyrazinamide susceptibility in Mycobacterium tuberculosis, mutant phenotypes, overview	Mycobacterium tuberculosis
additional information	catalytic Cys138	Mycobacterium tuberculosis