M-CSA Mechanism and Catalytic Site Atlas

Nudix hydrolase (YmfB)

Nudix hydrolases are enzymes that turn triphosphate nucleotides into monophosphate nucleotides in a stepwise manner which is crucial for the metabolism of thiamine pyrophosphate and important for several metabolic processes. In addition, they hydrolyse a wide range of organic pyrophosphates, including nucleotide sugars, dinucleoside polyphosphates, dinucleotide coenzymes and capped RNAs. They do this with a highly conserved 23-amino acid sequence called the Nudix motif. These proteins are present across the natural world from bacteria to humans.

Reference Protein and Structure

Sequence: A1AA28 (3.6.1.-) (Sequence Homologues) (PDB Homologues)
Biological species: Escherichia coli APEC O1 (Bacteria)
PDB: 3shd - Crystal structure of Nudix hydrolase Orf153, ymfB, from Escherichia coli K-1 (2.5 Å)
Catalytic CATH Domains: 3.90.79.10 (see all for 3shd)
Cofactors: Magnesium(2+) (2)

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Enzyme Reaction (EC:3.6.1.28)

water

CHEBI:15377

thiamine(1+) triphosphate(4-)

CHEBI:58938

→

hydrogenphosphate

CHEBI:43474

thiamine(1+) diphosphate(3-)

CHEBI:58937

hydron

CHEBI:15378

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: ThTPase,

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

Summary
Step 1
Step 2
Products
All Steps

Introduction

The divalent metal ions (here represented as Mg²⁺) lower the pKa of water to 5.2 and allow a hydroxide to form and attack a phosphate molecule. Glu23 and Arg119 stabilise the charges on the phosphates of the substrate. Although the EC:3.6.1.28 only shows one hydrolysis reaction (to form thiamine diphosphate) the mechanism depicted shows a second hydrolysis reaction to form thiamine monophosphate, which is the real product of this enzyme reaction.

Catalytic Residues Roles

UniProt	PDB* (3shd)
Glu55, Glu51	Glu55I, Glu51I	Glu51 and Glu55 coordinate the divalent metal ions into the active site. Mutation of these residues results in complete loss of function.	metal ligand
Glu23, Arg119	Glu23I, Arg119I	Glu23 and Arg119 are crucial for stabilising the negatively charged phosphates in the active site.	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, overall reactant used, hydrolysis, overall product formed

References

Hong M et al. (2014), Acta Crystallogr D Biol Crystallogr, 70, 1297-1310. Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) fromEscherichia coli. DOI:https://doi.org/10.1107/S1399004714002570.
Xu W et al. (2006), J Biol Chem, 281, 22794-22798. Three new Nudix hydrolases from Escherichia coli. DOI:10.1074/jbc.M603407200. PMID:16766526.

Step 1. The Mg²⁺ ions lower the pKa of water to allow a hydroxide to form and attack the terminal phosphate group. Glu23 and Arg119 stabilise the charges on the substrates phosphates.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Glu55I	metal ligand
Glu51I	metal ligand
Glu23I	electrostatic stabiliser
Arg119I	electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, hydrolysis

Step 2. A second hydroxide binds the two metal ion and the reaction is repeated, this time on the Beta phosphate, which leads to the formation of thiamine phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Glu23I	electrostatic stabiliser
Arg119I	electrostatic stabiliser
Glu55I	metal ligand
Glu51I	metal ligand

Chemical Components

ingold: bimolecular nucleophilic substitution, hydrolysis, overall product formed

Download: Image, Marvin File

Step 1. The Mg²⁺ ions lower the pKa of water to allow a hydroxide to form and attack the terminal phosphate group. Glu23 and Arg119 stabilise the charges on the substrates phosphates.

Step 2. A second hydroxide binds the two metal ion and the reaction is repeated, this time on the Beta phosphate, which leads to the formation of thiamine phosphate.

Products.

Contributors

Marko Babić, Antonio Ribeiro