Nicotinamidase

 

Pyrazinamide (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.

 

Reference Protein and Structure

Sequence
O58727 UniProt IPR000868 (Sequence Homologues) (PDB Homologues)
Biological species
Pyrococcus horikoshii OT3 (Bacteria) Uniprot
PDB
1im5 - Crystal Structure of Pyrazinamidase of Pyrococcus horikoshii in Complex with Zinc (1.65 Å) PDBe PDBsum 1im5
Catalytic CATH Domains
3.40.50.850 CATHdb (see all for 1im5)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.19)

nicotinamide
CHEBI:17154ChEBI
+
water
CHEBI:15377ChEBI
→
ammonium
CHEBI:28938ChEBI
+
nicotinate
CHEBI:32544ChEBI
Alternative enzyme names: YNDase, Nicotinamide amidase, Nicotinamide deaminase, Nicotine deamidase, NAMase,

Enzyme Mechanism

Introduction

The 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. Asp 10 deprotonates Cys 133, activating Cys as a nucleophile. The Cys 133 thiolate attacks the amide group of pyrazinamide. The tetrahedral intermediate is stabilised by an oxyanion hole comprising the backbone amides of Ala 129 and Cys 133. The tetrahedral state collapses to yield the acylated Cys 133. The NH2 leaving group is protonated by Asp 10 to yield ammonia. 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. 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.

Catalytic Residues Roles

UniProt PDB* (1im5)
Asp52, His54, His71 Asp52A, His54A, His71A Coordinates to Zinc ion metal ligand
Ser60, Glu101 Ser60A, Glu101A Stabilises water that are part of the Zinc coordination sphere electrostatic stabiliser
Lys94 Lys94A Lys 94 stabilises the negative charges on the Cys 133 thiolate and Asp 10 carboxylate, tuning their pKas by hydrogen bonding. electrostatic stabiliser
Cys133 (main-N), Ala129 (main-N) Cys133A (main-N), Ala129A (main-N) The backbone amide of Ala 129 forms part of the oxyanion hole. electrostatic stabiliser
Cys133 Cys133A Cys 133 is the nucleophilic cysteine which becomes acylated in the intermediate.
The backbone amide is part of the oxyanion hole.		 nucleofuge, nucleophile, proton acceptor, proton donor
Asp10 Asp10A Asp 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. proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, rate-determining step, unimolecular elimination by the conjugate base, heterolysis, intermediate collapse, overall product formed, enzyme-substrate complex cleavage, native state of enzyme regenerated

References

  1. Du X et al. (2001), Biochemistry, 40, 14166-14172. Crystal Structure and Mechanism of Catalysis of a Pyrazinamidase fromPyrococcus horikoshii†. DOI:10.1021/bi0115479. PMID:11714269.
  2. Sheng X et al. (2014), Org Biomol Chem, 12, 1265-1277. A QM/MM study of the catalytic mechanism of nicotinamidase. DOI:10.1039/C3OB42182A. PMID:24413890.
  3. Lemaitre N et al. (1999), Antimicrob Agents Chemother, 43, 1761-1763. 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. PMID:10390238.
  4. Romão MJ et al. (1992), J Mol Biol, 226, 1111-1130. Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2·0Åresolution. DOI:10.1016/0022-2836(92)91056-u. PMID:1381445.

Step 1. Asp10 deprotonates Cys133 which then nucleophilically attacks the carbon of the carbonyl group of Nicotinamide.

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Catalytic Residues Roles

Residue Roles
Ala129A (main-N) electrostatic stabiliser
Lys94A electrostatic stabiliser
Ser60A electrostatic stabiliser
Glu101A electrostatic stabiliser
Asp52A metal ligand
His54A metal ligand
His71A metal ligand
Cys133A (main-N) electrostatic stabiliser
Asp10A proton acceptor
Cys133A proton donor, nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, rate-determining step

Step 2. The amine group is protonated by Asp10 which initiates an elimination from the oxyanion which results in the cleavage of the amide bond an the release of ammonia.

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Catalytic Residues Roles

Residue Roles
Ser60A electrostatic stabiliser
Lys94A electrostatic stabiliser
Glu101A electrostatic stabiliser
Ala129A (main-N) electrostatic stabiliser
Cys133A (main-N) electrostatic stabiliser
Asp52A metal ligand
His54A metal ligand
His71A metal ligand
Asp10A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, heterolysis, intermediate collapse, overall product formed

Step 3. Asp10 deprotonates water which activates it to nucleophilically attack the carbon of the carbonyl group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser60A electrostatic stabiliser
Lys94A electrostatic stabiliser
Glu101A electrostatic stabiliser
Ala129A (main-N) electrostatic stabiliser
Cys133A (main-N) electrostatic stabiliser
Asp52A metal ligand
His54A metal ligand
His71A metal ligand
Asp10A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 4. The oxyanion initiates an elimination which results in the cleavage of the acyl-enzyme which releases Cys133 which then accepts a proton from Asp10 returning the active site to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser60A electrostatic stabiliser
Lys94A electrostatic stabiliser
Glu101A electrostatic stabiliser
Ala129A (main-N) electrostatic stabiliser
Cys133A (main-N) electrostatic stabiliser
Asp52A metal ligand
His54A metal ligand
His71A metal ligand
Cys133A nucleofuge
Asp10A proton donor
Cys133A proton acceptor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, enzyme-substrate complex cleavage, intermediate collapse, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Asp10 deprotonates Cys133 which then nucleophilically attacks the carbon of the carbonyl group of Nicotinamide.

Step 2. The amine group is protonated by Asp10 which initiates an elimination from the oxyanion which results in the cleavage of the amide bond an the release of ammonia.

Step 3. Asp10 deprotonates water which activates it to nucleophilically attack the carbon of the carbonyl group.

Step 4. The oxyanion initiates an elimination which results in the cleavage of the acyl-enzyme which releases Cys133 which then accepts a proton from Asp10 returning the active site to its native state.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, Charity Hornby