Hydroxymethylpyrimidine kinase

 

HMPP kinase is a key enzyme in the biosynthetic pathway for the formation of thiamine. The bacterial form of the enzyme described here is a member of the ribokinase/sugar kinase family, meaning that it displays significant sequence and structural homology with a large number of kinases with different substrates such as Adenosine kinase. It is unusual however because it is able to catalyse two consecutive steps in the synthesis of thiamine; phosphorylation of 4-amino-5-hydroxymethyl-5-methyl pyrimidine (HM) to give HMP followed by phosphorylation of HMP to give HMPP. This makes studying the enzyme interesting for understanding the specificity of the active site.

 

Reference Protein and Structure

Sequence
P55882 UniProt (2.7.1.49, 2.7.4.7) IPR004399 (Sequence Homologues) (PDB Homologues)
Biological species
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (Bacteria) Uniprot
PDB
1jxh - 4-Amino-5-hydroxymethyl-2-methylpyrimidine Phosphate Kinase from Salmonella typhimurium (2.3 Å) PDBe PDBsum 1jxh
Catalytic CATH Domains
3.40.1190.20 CATHdb (see all for 1jxh)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.7.1.49)

4-amino-5-hydroxymethyl-2-methylpyrimidine
CHEBI:16892ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
4-amino-2-methyl-5-phosphonatooxymethylpyrimidine(2-)
CHEBI:58354ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Hydroxymethylpyrimidine kinase (phosphorylating),

Enzyme Mechanism

Introduction

HMPP kinase catalyses an SN2 type in line displacement reaction where the substrate acts as the nucleophile to attack the gamma phosphate of ATP, forming a pentavalent phosphate transition state. This is stabilised by Mg2+and Lys 176 at the active site, and collapses to release the end products. Gly210, along with Thr211, Gly212 and Cys213 form the anion hole which also helps stabilise the transition state. A subsequent phosphorylation of the product HMP-P occurs by a similar in line displacement in an overlapping active site.

Catalytic Residues Roles

UniProt PDB* (1jxh)
Thr208, Thr208 (main-C) Thr208(230)A, Thr208(230)A (main-C) Thr208 through its carbonyl and side chain coordinates to Mg2+ ion that in turn stabilises the leaving group ADP. metal ligand
Thr211 (main-N), Gly212 (main-N), Cys213 Thr211(233)A (main-N), Gly212(234)A (main-N), Cys213(235)A Alongside Gly210, Thr211, Gly212 and Cys213 make up the anion hole in a conserved GTGC motif across the ribokinase family that is able to help stabilise the transition state. electrostatic stabiliser
Lys176 Lys176(198)A Acts to stabilise the transition state through electrostatic contacts with the gamma phosphate. electrostatic stabiliser
Gly210 (main-N) Gly210(232)A (main-N) Gly210 is apart of the anion hole and acts to stabilise the transition state through favourable contacts between the gamma phosphate and the amide group of the residue. electrostatic stabiliser
*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

References

  1. Cheng G et al. (2002), Structure, 10, 225-235. Crystal Structure of 4-Amino-5-Hydroxymethyl-2- Methylpyrimidine Phosphate Kinase from Salmonella typhimurium at 2.3 Å Resolution. DOI:10.1016/s0969-2126(02)00708-6. PMID:11839308.

Step 1.

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Catalytic Residues Roles

Residue Roles
Gly210(232)A (main-N) electrostatic stabiliser
Lys176(198)A electrostatic stabiliser
Thr208(230)A metal ligand
Thr208(230)A (main-C) metal ligand
Thr211(233)A (main-N) electrostatic stabiliser
Gly212(234)A (main-N) electrostatic stabiliser
Cys213(235)A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution

Step 1.

Contributors

Peter Sarkies, Gemma L. Holliday, Morwenna Hall