Inosine, adenosine, and guanine nucleoside hydrolase

 

Nucleoside hydrolases (NH's) are members of the purine salvage pathway that tend to scavenge purines from their environment.

The NH of of parasite, Trypanosoma vivax, catalyses the cleavage (hydrolysis) of the N-glycosidic bond between the anomeric carbon of ribose and the purine base in (specifically) inosine, adenosine, and guanine (an IAG-NH). This specificity is via an aromatic face-to-face stacking intercation, rather than general acid catalysis.

IAG-NH's represent potential targets for antiobiotic development and cancer therapy.

 

Reference Protein and Structure

Sequence
Q9GPQ4 UniProt IPR023186 (Sequence Homologues) (PDB Homologues)
Biological species
Trypanosoma vivax (Trypanosome) Uniprot
PDB
1hoz - CRYSTAL STRUCTURE OF AN INOSINE-ADENOSINE-GUANOSINE-PREFERRING NUCLEOSIDE HYDROLASE FROM TRYPANOSOMA VIVAX (1.6 Å) PDBe PDBsum 1hoz
Catalytic CATH Domains
3.90.245.10 CATHdb (see all for 1hoz)
Cofactors
Calcium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.2.2.1)

water
CHEBI:15377ChEBI
+
inosine
CHEBI:17596ChEBI
→
D-ribofuranose
CHEBI:47013ChEBI
+
hypoxanthine
CHEBI:17368ChEBI
Alternative enzyme names: N-ribosyl purine ribohydrolase, Nucleosidase, Nucleosidase g, Nucleoside hydrolase, Purine beta-ribosidase, Purine nucleoside hydrolase, Purine ribonucleosidase, Ribonucleoside hydrolase, N-D-ribosylpurine ribohydrolase, Inosine-adenosine-guanosine preferring nucleoside hydrolase, Purine-specific nucleoside N-ribohydrolase, IAG-nucleoside hydrolase, IAG-NH,

Enzyme Mechanism

Introduction

The catalytic reaction involves cleavage of the N-glycosidic bond between ribose and the purine base of the substrate. fast hydrolysis of the substrate scissile bond is followed by a slow, two-step release of ribose and a conformational change of the enzyme-ribose complex.

The enzyme binds to the pyridine base of the substrate between the aromatic side chains of Trp 83 and Trp 260, resulting in a face-to-face stacking arrangement. This raises the pKa of the aromatic substrate, and hence promotes protonation of the purine ring by elevating its basicity.

N7 protonation of the substrate from bulk solvent occurs since the solvent water is hydrogen bonded to the substrate N7. This results in quaternization of N in the substrate, and induces a positive charge in the purine ring, giving a chemically activated leaving group. Therefore the water molecule acts as a general acid to protonate the departing nucleobase anion. The N7-bonded water is in turn hydrogen bonded to waters in a channel.

The soft, electron rich indole ring of Trp 260 stabilises the positive charge on the N.

The activated purine ring is now a good leaving group. A ribooxocarbenium ion-like transition state is formed. The hydrolysis of the scissile bond is by a calcium-bound water molecule, Wat 1, which is hydrogen bonded to the general base, Asp 10, and the amide group of Asn 186.

The side chain of Asp 10 is hydrogen bonded to the nucleophilic water. This Asp 10 acts as a general base by abstracting a proton form Wat 1, activating it towards nucleophilic attack of the scissile bond. The Asn 186 side chain carbonyl stabilises the transition state by migrating closer to the O4' and its amide -NH hydrogen bonds to O3' of the substrate.

Cleavage of the N-glycosidic bond occurs, and there is a conformational change of the enzyme-ribose complex.

The ribose product is released from the active site, regenerating the enzyme.

Catalytic Residues Roles

UniProt PDB* (1hoz)
Asn186 Asn186(198)A Asn 186 hydrogen bonds to the nucleophilic Wat1, activating it towards nucleophilic attack of the scissile bond. The Asn 186 side chain carbonyl stabilises the transition state by migrating closer to the O4' and its amide -NH is hydrogen bonded to the O3'. activator, electrostatic stabiliser
Asp40 Asp40(52)A Asp 40 has some role in leaving group activation via interaction with the N3 of the 9-deazahypoxanthine ring activator
Trp260 Trp260(272)A Trp 260 is involved in pi-pi stacking to the substrate pyridine ring, raising its pKa, thus activating the pyridine ring towards protonation. The electron rich indole side chain of Trp 260 stabilises the positive charge on the pyridine N. activator, electrostatic stabiliser
Asp10 Asp10(22)A Is one of the catalytic Ca(II) ligands. Asp 10 acts as a general base. It activates Wat 1 to nucleophilic attack of the scissile bond by hydrogen bonding to the water molecule. proton shuttle (general acid/base), metal ligand
Asp261, Asp15, Thr137 Asp261(273)A, Asp15(27)A, Thr137(149)A Binds the catalytic Ca(II) ion. metal ligand
Trp83 Trp83(95)A Trp 83 is involved in pi-pi stacking to the substrate pyridine ring, raising its pKa, thus activating the pyridine ring towards protonation. activator
*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

References

  1. Chen N et al. (2015), J Chem Theory Comput, 11, 3180-3188. Mechanistic Insights into the Rate-Limiting Step in Purine-Specific Nucleoside Hydrolase. DOI:10.1021/acs.jctc.5b00045. PMID:26575755.
  2. Giannese F et al. (2013), Acta Crystallogr D Biol Crystallogr, 69, 1553-1566. Structures of purine nucleosidase fromTrypanosoma bruceibound to isozyme-specific trypanocidals and a novel metalorganic inhibitor. DOI:10.1107/s0907444913010792. PMID:23897478.
  3. Versées W et al. (2009), Biochim Biophys Acta, 1794, 953-960. Crystal structures of T. vivax nucleoside hydrolase in complex with new potent and specific inhibitors. DOI:10.1016/j.bbapap.2009.02.011. PMID:19281874.
  4. Versées W et al. (2006), J Mol Biol, 359, 331-346. Transition-state Complex of the Purine-specific Nucleoside Hydrolase of T.vivax: Enzyme Conformational Changes and Implications for Catalysis. DOI:10.1016/j.jmb.2006.03.026. PMID:16630632.
  5. Mazumder-Shivakumar D et al. (2005), Biochemistry, 44, 7805-7817. Computational Study of IAG-Nucleoside Hydrolase:  Determination of the Preferred Ground State Conformation and the Role of Active Site Residues†. DOI:10.1021/bi047394h. PMID:15909995.
  6. Versées W et al. (2004), J Mol Biol, 338, 1-6. Leaving Group Activation by Aromatic Stacking: An Alternative to General Acid Catalysis. DOI:10.1016/j.jmb.2004.02.049. PMID:15050818.
  7. Versées W et al. (2002), J Biol Chem, 277, 15938-15946. Enzyme-Substrate Interactions in the Purine-specific Nucleoside Hydrolase from Trypanosoma vivax. DOI:10.1074/jbc.m111735200. PMID:11854281.
  8. Versées W et al. (2001), J Mol Biol, 307, 1363-1379. Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax11Edited by R. Huber. DOI:10.1006/jmbi.2001.4548. PMID:11292348.

Step 1.

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Catalytic Residues Roles

Residue Roles
Trp83(95)A activator
Asn186(198)A activator, electrostatic stabiliser
Trp260(272)A activator, electrostatic stabiliser
Asp10(22)A proton shuttle (general acid/base), metal ligand
Asp15(27)A metal ligand
Thr137(149)A metal ligand
Asp261(273)A metal ligand
Asp40(52)A activator

Chemical Components

Step 1.

Contributors

Gemma L. Holliday, Emma Penn