Orotate phosphoribosyltransferase

 

Orotate phosphoribosyltransferase (OPRTase) is involved in the biosynthesis of pyrimidine nucleotides. In the pyrimidine synthesis pathway, OPRT catalyses the reversible phosphoribosyl transfer from 5'-phospho-alpha-D-ribose 1'-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5'-monophosphate (OMP).

The mechanism of PRTases has been the subject of much debate over the years. However, stereochemical results argue against mechanisms which invoke a covalent enzyme-phosphoribosyl intermediate. Kinetic studies argue against a direct SN2-type displacement. Thus, a two-step, SN1-type mechanisms with oxocarbonium-like transition states or intermediates has been proposed as the most likely mechanism. However, there is still much debate as to the exact roles of the residues involved.

 

Reference Protein and Structure

Sequence
P0A7E3 UniProt (2.4.2.10) IPR023031 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1oro - A FLEXIBLE LOOP AT THE DIMER INTERFACE IS A PART OF THE ACTIVE SITE OF THE ADJACENT MONOMER OF ESCHERICHIA COLI OROTATE PHOSPHORIBOSYLTRANSFERASE (2.4 Å) PDBe PDBsum 1oro
Catalytic CATH Domains
3.40.50.2020 CATHdb (see all for 1oro)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.10)

5-O-phosphonato-alpha-D-ribofuranosyl diphosphate(5-)
CHEBI:58017ChEBI
+
orotate
CHEBI:30839ChEBI
→
orotidine 5'-phosphate(3-)
CHEBI:57538ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: OPRT, OPRTase, Orotate phosphoribosyl pyrophosphate transferase, Orotic acid phosphoribosyltransferase, Orotidine 5'-monophosphate pyrophosphorylase, Orotidine monophosphate pyrophosphorylase, Orotidine phosphoribosyltransferase, Orotidine-5'-phosphate pyrophosphorylase, Orotidylate phosphoribosyltransferase, Orotidylate pyrophosphorylase, Orotidylic acid phosphorylase, Orotidylic acid pyrophosphorylase, Orotidylic phosphorylase, Orotidylic pyrophosphorylase, Orotidine-5'-phosphate diphosphorylase,

Enzyme Mechanism

Introduction

Although there has been much debate as to the exact mechanism of this protein, it is now thought to proceed via an SN1-type mechanism. Here, the phosphate group (activated by the divalent metal ion) dissociates, forming an oxycarbenium intermediate which is then attacked by the orotate substrate to form the OMP product.

Catalytic Residues Roles

UniProt PDB* (1oro)
His105 His105A Thought to play a direct role for His105 in binding to the alpha-phosphate, may also play a role in protonation or deprotonation of the leaving or attacking pyrophosphate, respectively. electrostatic stabiliser
Lys103 Lys103A Thought to act as a general acid/base. Lys103 appears poised to provide geometric stabilisation, proton transfer, and/or charge neutralisation at the transition state. proton acceptor, electrostatic stabiliser, 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

unimolecular elimination by the conjugate base, overall reactant used, bimolecular nucleophilic addition, proton transfer, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. González-Segura L et al. (2007), Biochemistry, 46, 14075-14086. Ternary complex formation and induced asymmetry in orotate phosphoribosyltransferase. DOI:10.1021/bi701023z. PMID:18020427.
  2. Donini S et al. (2017), Sci Rep, 7, 1180-. Structural investigations on orotate phosphoribosyltransferase from Mycobacterium tuberculosis, a key enzyme of the de novo pyrimidine biosynthesis. DOI:10.1038/s41598-017-01057-z. PMID:28446777.
  3. Wang GP et al. (2012), Biochemistry, 51, 4406-4415. Loop residues and catalysis in OMP synthase. DOI:10.1021/bi300082s. PMID:22531099.
  4. Grubmeyer C et al. (2012), Biochemistry, 51, 4397-4405. Structure of Salmonella typhimurium OMP synthase in a complete substrate complex. DOI:10.1021/bi300083p. PMID:22531064.
  5. Zhang Y et al. (2010), J Am Chem Soc, 132, 17023-17031. Leaving group activation and pyrophosphate ionic state at the catalytic site of Plasmodium falciparum orotate phosphoribosyltransferase. DOI:10.1021/ja107806j. PMID:21067187.
  6. Zhang Y et al. (2010), J Am Chem Soc, 132, 8787-8794. Pyrophosphate interactions at the transition states of Plasmodium falciparum and human orotate phosphoribosyltransferases. DOI:10.1021/ja102849w. PMID:20527751.
  7. Zhang Y et al. (2009), J Am Chem Soc, 131, 4685-4694. Transition states of Plasmodium falciparum and human orotate phosphoribosyltransferases. DOI:10.1021/ja808346y. PMID:19292447.
  8. Wang GP et al. (1999), Biochemistry, 38, 284-295. Motional dynamics of the catalytic loop in OMP synthase. DOI:10.1021/bi982057s. PMID:9890909.
  9. Tao W et al. (1996), Biochemistry, 35, 14-21. Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase. DOI:10.1021/bi951898l. PMID:8555167.
  10. Ozturk DH et al. (1995), Biochemistry, 34, 10755-10763. Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase. PMID:7545005.
  11. Scapin G et al. (1995), Biochemistry, 34, 10744-10754. The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate. PMID:7545004.
  12. Bhatia MB et al. (1993), Arch Biochem Biophys, 303, 321-325. The role of divalent magnesium in activating the reaction catalyzed by orotate phosphoribosyltransferase. DOI:10.1006/abbi.1993.1290. PMID:7685580.

Step 1. The diphosphate group dissociates from the PRPP substrate, forming an oxycarbenium ion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys103A electrostatic stabiliser
His105A electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall reactant used

Step 2. Lys103 (or a bound water molecule, activated by Lys103) abstracts a proton from the substrate which initiates the nucleophilic attack on the oxycarmenium ion, forming the final products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His105A electrostatic stabiliser
Lys103A proton acceptor

Chemical Components

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

Step 3. Inferred return step in which water (although it could also be the product diphosphate) abstracts the proton from Lys103.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys103A proton donor

Chemical Components

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

Step 1. The diphosphate group dissociates from the PRPP substrate, forming an oxycarbenium ion intermediate.

Step 2. Lys103 (or a bound water molecule, activated by Lys103) abstracts a proton from the substrate which initiates the nucleophilic attack on the oxycarmenium ion, forming the final products.

Step 3. Inferred return step in which water (although it could also be the product diphosphate) abstracts the proton from Lys103.

Products.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday