5'-nucleotidase (bacterial)

 

5'-nucleotidase is a zinc-containing enzyme involved in ATP hydrolysis and is bound to the membrane as an extracellular nucleotidase, or ectonucleotidase. Bacterial 5'-nucleotidases show a significant sequence homology to animal counterparts suggesting a common evolutionary origin. It is also related to 2',3'-cyclic phosphodiesterases and apyrases, all being part of the superfamily of metallophosphoesterases which also includes the Ser/Thr protein phosphatases and the purple acid phosphatases. The enzyme also has a UDP-sugar hydrolase activity which catalyses the periplasmic degradation of external UDP-glucose to uridine, glucose-1-phosphate and phosphate.

 

Reference Protein and Structure

Sequence
P07024 UniProt (3.1.3.5, 3.6.1.45) IPR006179 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1ush - 5'-NUCLEOTIDASE FROM E. COLI (1.73 Å) PDBe PDBsum 1ush
Catalytic CATH Domains
3.60.21.10 CATHdb 3.90.780.10 CATHdb (see all for 1ush)
Cofactors
Zinc(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.1.3.5)

water
CHEBI:15377ChEBI
+
nucleoside 5'-monophosphate(2-)
CHEBI:58043ChEBI
→
hydrogenphosphate
CHEBI:43474ChEBI
+
ribonucleoside
CHEBI:18254ChEBI
Alternative enzyme names: 5'-AMP nucleotidase, 5'-AMPase, 5'-adenylic phosphatase, 5'-mononucleotidase, AMP phosphatase, AMP phosphohydrolase, AMPase, IMP 5'-nucleotidase, UMPase, Adenosine 5'-phosphatase, Adenosine monophosphatase, Snake venom 5'-nucleotidase, Thimidine monophosphate nucleotidase, Uridine 5'-nucleotidase,

Enzyme Mechanism

Introduction

5'-nucleotidase catalyses the hydrolytic cleavage of 5'-mononucleotides to nucleoside and phosphate by nucleophilic attack of a water molecule on the phosphorous. A water ligand is coordinated to the di-zinc centre. The phosphate group of the substrate binds with one oxygen atom to a metal ion. The metal-bound water attacks the phosphorous nucleophilically, with the proton transferred to His 117. The pentahedral transition state is stabilised by both metal ions, Asn 116, Arg 375, Arg 379, Arg 410 and His 117. After expulsion of the leaving group the tetrahedral phosphate ion remains bound to the dimetal centre.

Catalytic Residues Roles

UniProt PDB* (1ush)
Asn116 Asn116A Stabilises the pentacoordinate transition state. metal ligand, electrostatic stabiliser
Asp41, Asp84, His217, His252, Gln254, His43, Asn116 Asp41A, Asp84A, His217A, His252A, Gln254A, His43A, Asn116A Coordinate the metal ions. metal ligand
His117 His117A Facilitates proton transfer from water and acts to stabilise the pentacoordinate transition state. proton acceptor, increase nucleophilicity, metal ligand, electrostatic stabiliser
Asp120 Asp120A Activates the His 117 to allow it to act as a general base. increase basicity, electrostatic stabiliser
Arg375 Arg375A Contributes to positive potential at the active site and stabilise transition state. increase electrophilicity, electrostatic stabiliser, increase acidity
Arg379, Arg375, Arg410 Arg379A, Arg375A, Arg410A Contributes to the distinct positive potential at the active site and stabilises the pentacoordinate transition state. increase electrophilicity, electrostatic stabiliser, increase acidity
*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

overall reactant used, proton transfer, bimolecular nucleophilic addition, heterolysis, overall product formed, decoordination from a metal ion, coordination to a metal ion

References

  1. Knöfel T et al. (2001), J Mol Biol, 309, 239-254. Mechanism of hydrolysis of phosphate esters by the dimetal center of 5′-nucleotidase based on crystal structures. DOI:10.1006/jmbi.2001.4656. PMID:11491293.
  2. Mitić N et al. (2006), Chem Rev, 106, 3338-3363. The catalytic mechanisms of binuclear metallohydrolases. DOI:10.1021/cr050318f. PMID:16895331.
  3. Knöfel T et al. (1999), Nat Struct Biol, 6, 448-453. X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site. DOI:10.1038/8253. PMID:10331872.

Step 1. His117/Asp120 dyad and the two zinc ions activate a water molecule to perform a nucleophiliic attack on the phosphate group. This forms a penta-covalent intermediate stabilized by the arginine residues. The bridging water is thought to be the nucleophile as it has the lowest pKa out of the potential water molecules.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg375A electrostatic stabiliser
Asp120A electrostatic stabiliser
Arg410A electrostatic stabiliser
Arg379A electrostatic stabiliser
Asp41A metal ligand
His43A metal ligand
Asp84A metal ligand
Asn116A metal ligand
His117A metal ligand
His217A metal ligand
His252A metal ligand
Gln254A metal ligand
Asp120A increase basicity
His117A increase nucleophilicity
Arg375A increase electrophilicity
Arg379A increase electrophilicity
Arg410A increase electrophilicity
Asn116A electrostatic stabiliser
His117A electrostatic stabiliser, proton acceptor

Chemical Components

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

Step 2. Since no acidic residues are positioned to protonate the leaving group this role is thought to be performed by a solvent water molecule. This leaves a tetrahedral phosphate bound to the zinc ions.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg375A electrostatic stabiliser
Arg379A electrostatic stabiliser
Arg410A electrostatic stabiliser
Asp41A metal ligand
His43A metal ligand
Asp84A metal ligand
Asn116A metal ligand
His217A metal ligand
His252A metal ligand
Gln254A metal ligand
Arg375A increase acidity
Arg379A increase acidity
Arg410A increase acidity
Asn116A electrostatic stabiliser
His117A electrostatic stabiliser

Chemical Components

heterolysis, proton transfer, overall product formed

Step 3. Phosphate leaves the active site and the active site transitions to its resting state with each zinc ion being 5 coordinate and no terminal water molecules coordianted.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp41A metal ligand
His43A metal ligand
Asp84A metal ligand
Asn116A metal ligand
His217A metal ligand
His252A metal ligand
Gln254A metal ligand
Arg375A electrostatic stabiliser
Arg379A electrostatic stabiliser
Arg410A electrostatic stabiliser
Asn116A electrostatic stabiliser
His117A electrostatic stabiliser

Chemical Components

decoordination from a metal ion, coordination to a metal ion, overall product formed
Download: Image, Marvin File

Step 1. His117/Asp120 dyad and the two zinc ions activate a water molecule to perform a nucleophiliic attack on the phosphate group. This forms a penta-covalent intermediate stabilized by the arginine residues. The bridging water is thought to be the nucleophile as it has the lowest pKa out of the potential water molecules.

Step 2. Since no acidic residues are positioned to protonate the leaving group this role is thought to be performed by a solvent water molecule. This leaves a tetrahedral phosphate bound to the zinc ions.

Step 3. Phosphate leaves the active site and the active site transitions to its resting state with each zinc ion being 5 coordinate and no terminal water molecules coordianted.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, James Willey