UDP-glucose 4-epimerase

 

UDP-glucose 4-epimerase (EC:5.1.3.2) interconverts UDP-glucose and UDP-galactose which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) via the transient reduction of NAD.

 

Reference Protein and Structure

Sequence
P09147 UniProt (5.1.3.2) IPR005886 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1xel - UDP-GALACTOSE 4-EPIMERASE FROM ESCHERICHIA COLI (1.8 Å) PDBe PDBsum 1xel
Catalytic CATH Domains
3.40.50.720 CATHdb (see all for 1xel)
Cofactors
Nadph(4-) (1)
Click To Show Structure

Enzyme Reaction (EC:5.1.3.2)

UDP-alpha-D-galactose(2-)
CHEBI:66914ChEBI
→
UDP-alpha-D-glucose(2-)
CHEBI:58885ChEBI
Alternative enzyme names: 4-epimerase, UDP-D-galactose 4-epimerase, UDP-galactose 4-epimerase, UDP-glucose epimerase, UDPG-4-epimerase, UDPgalactose 4-epimerase, UDPglucose 4-epimerase, Galactowaldenase, Uridine diphosphate galactose 4-epimerase, Uridine diphosphate glucose 4-epimerase, Uridine diphospho-galactose-4-epimerase, Uridine diphosphoglucose 4-epimerase, Uridine diphosphoglucose epimerase,

Enzyme Mechanism

Introduction

Tyr149 initiates the reaction via the abstraction of a proton from the substrate, which results in a hydride being transferred to the NAD cofactor. The intermediate then undergoes a conformational change in the active site. The rate-determining step of this reaction is the conformational change induced upon binding of the substrate. This is due to the fact that the mutation Tyr149Phe decreases the rate of the hydride transfer and the rate-determining conformational change by the same degree. In the final step, the hydride is returned and Tyr149 deprotonated.

Catalytic Residues Roles

UniProt PDB* (1xel)
Ser124 Ser124A Helps stabilise and activate the tyrosine residue. activator, hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Tyr149 Tyr149A Acts as a general acid/base, initiating the hydride transfer from the substrate to the NAD coactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys153 Lys153A Activates the NAD cofactor in the reverse of the initial hydride transfer. activator, hydrogen bond 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

bimolecular elimination, hydride transfer, aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation, aromatic unimolecular elimination by the conjugate base, bimolecular nucleophilic addition, proton transfer, intermediate terminated, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated

References

  1. Berger E et al. (2001), Biochemistry, 40, 6699-6705. Acid−Base Catalysis by UDP-Galactose 4-Epimerase:  Correlations of Kinetically Measured Acid Dissociation Constants with Thermodynamic Values for Tyrosine 149†. DOI:10.1021/bi0104571. PMID:11380265.

Step 1. Tyr149 deprotonates the 4-hydroxide of the substrate, resulting in an elimination of the hydride from the C4 position. The hydride then attacks the NAD cofactor in a nucleophilic addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys153A hydrogen bond donor
Tyr149A hydrogen bond acceptor
Ser124A hydrogen bond donor, hydrogen bond acceptor, electrostatic stabiliser
Tyr149A proton acceptor

Chemical Components

ingold: bimolecular elimination, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation

Step 2. The 4-ketohexopyranosyl ring of the intermediate rotates to place opposite faces of the 4-keto group in position for hydride from NADH. This conformational change potentates non-stereospecific hydride transfer, an essential mechanistic process in epimerisation [PMID:11380265]. The hydride transfer initiates the deprotonation of Tyr149.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys153A hydrogen bond donor, activator
Tyr149A hydrogen bond donor, hydrogen bond acceptor
Ser124A hydrogen bond donor, hydrogen bond acceptor, activator
Tyr149A proton donor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, ingold: bimolecular nucleophilic addition, hydride transfer, proton transfer, intermediate terminated, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Tyr149 deprotonates the 4-hydroxide of the substrate, resulting in an elimination of the hydride from the C4 position. The hydride then attacks the NAD cofactor in a nucleophilic addition reaction.

Step 2. The 4-ketohexopyranosyl ring of the intermediate rotates to place opposite faces of the 4-keto group in position for hydride from NADH. This conformational change potentates non-stereospecific hydride transfer, an essential mechanistic process in epimerisation [PMID:11380265]. The hydride transfer initiates the deprotonation of Tyr149.

Products.

Introduction

An alternate mechanism to the first one, where serine is directly involved in proton transfer acting as a proton relay, this is mechanism is thought to be more consistent with the E.coli enzyme while the first mechanism is more applicable to the human enzyme. The remainder of the mechanism is consistent with the first one.

Catalytic Residues Roles

UniProt PDB* (1xel)
Ser124 Ser124A Acts as a proton relay to initiate hydride transfer to the cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay, electrostatic stabiliser
Tyr149 Tyr149A Acts as a general acid/base, initiating the hydride transfer from the substrate to the coactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys153 Lys153A Activates the NAD cofactor in the reverse of the initial hydride transfer activator, hydrogen bond 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

bimolecular elimination, hydride transfer, bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation, proton relay, overall product formed, native state of enzyme regenerated, aromatic unimolecular elimination by the conjugate base, intermediate terminated, native state of cofactor regenerated

References

  1. Frey PA et al. (2013), Acc Chem Res, 46, 1417-1426. Chemical and stereochemical actions of UDP-galactose 4-epimerase. DOI:10.1021/ar300246k. PMID:23339688.

Step 1. In this alternate mechanism Tyr149 deprotonates Ser124 which simultaneously deprotonates the 4-hydroxide of the substrate, resulting in an elimination of the hydride from the C4 position. The hydride then attacks the NAD cofactor in a nucleophilic addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser124A proton relay
Ser124A hydrogen bond donor
Lys153A hydrogen bond donor
Ser124A hydrogen bond acceptor
Tyr149A hydrogen bond acceptor
Ser124A electrostatic stabiliser
Ser124A proton donor
Tyr149A proton acceptor
Ser124A proton acceptor

Chemical Components

ingold: bimolecular elimination, hydride transfer, ingold: bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation, proton relay

Step 2. The 4-ketohexopyranosyl ring of the intermediate rotates to place opposite faces of the 4-keto group in position for hydride from NADH. This conformational change potentates non-stereospecific hydride transfer, an essential mechanistic process in epimerisation. The hydride transfer intiates the proton relay from the product via Ser 124 to Tyr 149.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys153A hydrogen bond donor, activator
Ser124A hydrogen bond donor
Tyr149A hydrogen bond donor
Ser124A hydrogen bond acceptor
Tyr149A hydrogen bond acceptor
Ser124A proton relay, proton acceptor
Tyr149A proton donor
Ser124A proton donor

Chemical Components

proton relay, overall product formed, native state of enzyme regenerated, ingold: aromatic unimolecular elimination by the conjugate base, ingold: bimolecular nucleophilic addition, hydride transfer, intermediate terminated, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. In this alternate mechanism Tyr149 deprotonates Ser124 which simultaneously deprotonates the 4-hydroxide of the substrate, resulting in an elimination of the hydride from the C4 position. The hydride then attacks the NAD cofactor in a nucleophilic addition reaction.

Step 2. The 4-ketohexopyranosyl ring of the intermediate rotates to place opposite faces of the 4-keto group in position for hydride from NADH. This conformational change potentates non-stereospecific hydride transfer, an essential mechanistic process in epimerisation. The hydride transfer intiates the proton relay from the product via Ser 124 to Tyr 149.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, James Willey