Inosine-uridine preferring nucleoside hydrolase

 

Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-NH). The enzyme is homotetrameric (4 x 34 kDa subunits) and exhibits no known allosteric properties.

N-ribosyl hydrolase catalyses the hydrolysis of carbon-nitrogen bonds in all commonly occurring purine and pyrimidine nucleosides via an oxocarbenium-ion transition state. The hydrolysis forms ribose and the associated base. The enzyme has a preference for inosine and uridine as substrates. The substrate specificity and kinetic constants are consistent with His241 acting as a proton donor to activate the hypoxanthine leaving group. Although the enzyme is established as a member of the nonspecific nucleoside hydrolases, it has a unique substrate specificity. Its mechanism includes a tightly bound catalytic Ca(II).

The enzyme is involved in the purine salvage pathways of protozoan parasites and has not been found in mammals, since mammals possess an endogenous biochemical pathway which releases nucleosides by phospholysis, catalysed by nucleoside phosphorylase. Protozoan parasites lack de novo purine biosynthesis pathways and are dependent upon exogenous purines, salvaged from a host organism.

 

Reference Protein and Structure

Sequence
Q27546 UniProt (3.2.2.2, 3.2.2.3) IPR023186 (Sequence Homologues) (PDB Homologues)
Biological species
Crithidia fasciculata (Parasitic excavate) Uniprot
PDB
2mas - PURINE NUCLEOSIDE HYDROLASE WITH A TRANSITION STATE INHIBITOR (2.3 Å) PDBe PDBsum 2mas
Catalytic CATH Domains
3.90.245.10 CATHdb (see all for 2mas)
Cofactors
Calcium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.2.2.1)

nebularine
CHEBI:18255ChEBI
+
water
CHEBI:15377ChEBI
→
7H-purine
CHEBI:17258ChEBI
+
D-ribofuranose
CHEBI:47013ChEBI
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 proposed active centre is a relatively large cavity. The cavity is roughly cylindrical, located at the region identified as the "topological switchpoint". From modelling studies, PHE 167 seems to be in proper orientation to interact with the purine substrate in a base-stacking interaction, as typically observed in proteins which bind nucleotides. The carboxylate groups of three of four aspartate residues found in the cavity (ASP 10, ASP 14, ASP15 and ASP 242 - ASP 14 does not coordinate) along with THR 126, coordinate a divalent cation (possibly calcium).

A proposed mechanism suggests ribooxocarbenium stabilisation with weak leaving group activation. His 241 acts as an acid to protonate the N7 of the leaving purine. Ca(II) ion together with Asp10 activates a water molecule which nucleophilically attacks ribose C1'. Ca(II) ion serves to decrease the pKa of the attacking water molecule while Asp10 accept a proton from the water molecule. These leads to a ribooxocarbenium transition state which will spontaneously dissociate to form ribose and purine. The transition state is stabilised by Asn168, which electrostatically interacts with charged ribose O4'.

Catalytic Residues Roles

UniProt PDB* (2mas)
Asp10 Asp10(9)A Acts as an acid to deprotonate a water molecule, which acts as a nucleophile to attack ribose C1'. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor
Asn168 Asn168(167)A Stabilises the transition state by electrostatically interacts with charged ribose O4'. hydrogen bond donor, electrostatic stabiliser
His241 His241(240)A Activates the leaving group by donating a proton to purine N7. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Phe167 Phe167(166)A Interacts with the purine substrate in a base-stacking interaction, helping to stabilise the intermediates and also effects substrate specificity. electrostatic stabiliser
Asp15, Asp10, Thr126 (main-C), Asn39, Asp242 Asp15(14)A, Asp10(9)A, Thr126(125)A (main-C), Asn39(38)A, Asp242(241)A Forms part of the Ca(II) binding site. metal ligand
*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

bimolecular nucleophilic substitution, overall reactant used, overall product formed, hydrolysis, proton transfer, native state of enzyme regenerated, inferred reaction step

References

  1. Degano M et al. (1998), Biochemistry, 37, 6277-6285. Trypanosomal Nucleoside Hydrolase. A Novel Mechanism from the Structure with a Transition-State Inhibitor†. DOI:10.1021/bi973012e. PMID:9572842.
  2. Saen-Oon S et al. (2008), Biophys J, 94, 4078-4088. Remote Mutations and Active Site Dynamics Correlate with Catalytic Properties of Purine Nucleoside Phosphorylase. DOI:10.1529/biophysj.107.121913. PMID:18234834.
  3. Mazumder D et al. (2002), J Am Chem Soc, 124, 8825-8833. Computer Simulations of Trypanosomal Nucleoside Hydrolase:  Determination of the Protonation State of the Bound Transition-State Analogue. DOI:10.1021/ja020312x. PMID:12137535.
  4. Shi W et al. (1999), J Biol Chem, 274, 21114-21120. Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-å crystal structure. PMID:10409664.
  5. Gopaul DN et al. (1996), Biochemistry, 35, 5963-5970. Inosine−Uridine Nucleoside Hydrolase fromCrithidia fasciculata. Genetic Characterization, Crystallization, and Identification of Histidine 241 as a Catalytic Site Residue†,‡. DOI:10.1021/bi952998u. PMID:8634237.
  6. Degano M et al. (1996), Biochemistry, 35, 5971-5981. Three-Dimensional Structure of the Inosine−Uridine NucleosideN-Ribohydrolase fromCrithidiafasciculata†,‡. DOI:10.1021/bi952999m. PMID:8634238.

Step 1. Asp10 deprotonates water, which initiates a nucleophilic attack on the C1 of the ribose ring in a substitution reaction which eliminates the purine with concomitant deprotonation of His241.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn168(167)A hydrogen bond donor, electrostatic stabiliser
His241(240)A hydrogen bond donor
Asp10(9)A hydrogen bond acceptor
Asp10(9)A metal ligand
Asp15(14)A metal ligand
Asp242(241)A metal ligand
Asn39(38)A metal ligand
Thr126(125)A (main-C) metal ligand
Phe167(166)A electrostatic stabiliser
His241(240)A proton donor
Asp10(9)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, hydrolysis, proton transfer

Step 2. His241 deprotonates water and water deprotonates Asp10 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His241(240)A hydrogen bond acceptor
Asp10(9)A hydrogen bond donor
Asp10(9)A metal ligand
Asp15(14)A metal ligand
Asp242(241)A metal ligand
Asn39(38)A metal ligand
Thr126(125)A (main-C) metal ligand
His241(240)A proton acceptor
Asp10(9)A proton donor

Chemical Components

native state of enzyme regenerated, inferred reaction step, proton transfer
Download: Image, Marvin File

Step 1. Asp10 deprotonates water, which initiates a nucleophilic attack on the C1 of the ribose ring in a substitution reaction which eliminates the purine with concomitant deprotonation of His241.

Step 2. His241 deprotonates water and water deprotonates Asp10 in an inferred return step.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Anna Waters, Craig Porter