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Catalytic Site Atlas

CSA LITERATURE entry for 1im5

E.C. namenicotinamidase
SpeciesPyrococcus horikoshii (Bacteria)
E.C. Number (IntEnz) 3.5.1.19
CSA Homologues of 1im5
CSA Entries With UniProtID
CSA Entries With EC Number 3.5.1.19
PDBe Entry 1im5
PDBSum Entry 1im5
MACiE Entry 1im5

Literature Report

IntroductionPyrazinamide (PZA) is a prodrug used in the treatment of tuberculosis. PZA is converted to the active pyrazinoic acid by the bacterial PZAase enzyme; mutations in PZAase can therefore confer PZA resistance to M. tuberculosis.
The gene product of Pyrococcus horikoshii 999 (PH999) shows PZAase and nicotinamidase activity, and extensive sequence homology to M. tuberculosis PZAase.
MechansimThe mechanism of PH999 can be inferred by homology to other enzymes like Arthrobacter sp. CSHase and E. coli YcaC. The mechanism of substrate amide hydrolysis is likely to be via an acylated cysteine intermediate.
1) Asp 10 deprotonates Cys 133, activating Cys as a nucleophile. 2) The Cys 133 thiolate attacks the amide group of pyrazinamide. 3) The tetrahedral intermediate is stabilised by an oxyanion hole comprising the backbone amides of Ala 129 and Cys 133. 4) The tetrahedral state collapses to yield the acylated Cys 133. The NH2 leaving group is protonated by Asp 10 to yield ammonia. 5) Zn(II) coordinates a hydroxide ion that attacks the newly formed thioester. 6) The tetrahedral intermediate is again stabilised by the oxyanion hole. 7) A water molecule binds to Zn(II); Zn(II) acidifies the water so that Asp 10 can deprotonate it. (The resulting hydroxide ion attacks the acyl-enzyme in the next catalytic cycle.) 8) The tetrahedral intermediate collapses. Asp 10 protonates the leaving Cys 133 thiolate.
Lys 94 is likely to stabilise any charge on Cys 133 and Asp 10 during the reaction.
Reaction

Catalytic Sites for 1im5

Annotated By Reference To The Literature - Site 2 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
AspA1010macie:sideChainAsp 10 deprotonates Cys 133 and water, both of which act as nucleophiles during the reaction. Asp 10 transfers this proton to the leaving group, i.e. the Cys 133 thiolate, and -NH2 to give ammonia.
CysA133133macie:sideChainCys 133 is the nucleophilic cysteine which becomes acylated in the intermediate.
The backbone amide is part of the oxyanion hole.
CysA133133macie:mainChainAmideCys 133 is the nucleophilic cysteine which becomes acylated in the intermediate.
The backbone amide is part of the oxyanion hole.
AlaA129129macie:mainChainAmideThe backbone amide of Ala 129 forms part of the oxyanion hole.
LysA9494macie:sideChainLys 94 stabilises the negative charges on the Cys 133 thiolate and Asp 10 carboxylate, tuning their pKas by hydrogen bonding.

Literature References

Notes:
Du X
Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii.
Biochemistry 2001 40 14166-14172
PubMed: 11714269
Romão MJ
Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 A resolution.
J Mol Biol 1992 226 1111-1130
PubMed: 1381445
Lemaitre N
Characterization of new mutations in pyrazinamide-resistant strains of Mycobacterium tuberculosis and identification of conserved regions important for the catalytic activity of the pyrazinamidase PncA.
Antimicrob Agents Chemother 1999 43 1761-1763
PubMed: 10390238
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