UDP-glucose-hexose-1-phosphate uridylyltransferase

 

Nucleotidyltransferases catalyse the covalent modification of a variety of biological molecules. These reactions are crucial for the synthesis of coenzymes, cyclic nucleotides, polynucleotides, and nucleotide sugars. These reactions involve substitutions at the R -phosphorus of a nucleotidyl donor substrate and result in displacement of a phosphoryl ester or pyrophosphate. Substrates for such reactions may include nucleoside di- or triphosphates, as well as nucleotide sugars, such as UDP-Glc. Galactose-1-phosphate uridylyltransferase (hexose-1-phosphate uridylyltransferase) catalyses the exchange of the UMP moiety between the hexose 1-phosphates of Glc and Gal and their corresponding UDP-sugar. The enzyme is distinct among nucleotidyl transferases that use phosphates as acceptor groups in that it is the only one that does not utilise nucleoside di- or triphosphates as the nucleotidyl donor substrate. The reaction is part of the Leloir pathway of galactose metabolism required for the normal equilibration of UDP-hexoses among most organisms. Deficiencies in uridylyltransferase activity culminate in the metabolic disease galactosemia, which occurs as an autosomal recessive trait.

 

Reference Protein and Structure

Sequence
P09148 UniProt (2.7.7.12) IPR001937 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1hxq - THE STRUCTURE OF NUCLEOTIDYLATED GALACTOSE-1-PHOSPHATE URIDYLYLTRANSFERASE FROM ESCHERICHIA COLI AT 1.86 ANGSTROMS RESOLUTION (1.86 Å) PDBe PDBsum 1hxq
Catalytic CATH Domains
3.30.428.10 CATHdb (see all for 1hxq)
Cofactors
Zinc(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.7.7.12)

alpha-D-galactose 1-phosphate(2-)
CHEBI:58336ChEBI
+
UDP-alpha-D-glucose(2-)
CHEBI:58885ChEBI
→
alpha-D-glucose 1-phosphate(2-)
CHEBI:58601ChEBI
+
UDP-alpha-D-galactose(2-)
CHEBI:66914ChEBI
Alternative enzyme names: UDPglucose:alpha-D-galactose-1-phosphate uridylyltransferase, UDPglucose--hexose-1-phosphate uridylyltransferase, Hexose 1-phosphate uridyltransferase, Hexose-1-phosphate uridylyltransferase, Uridyl transferase, Uridyltransferase, Uridylyl removing enzyme, Galactose-1-phosphate uridylyltransferase, Gal-1-P uridylyltransferase, UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase,

Enzyme Mechanism

Introduction

His166 in the active site of the the enzyme in Escherichia coli attacks the alpha-phosphorus of UDP--Glc, displaces Glc-1-P, and forms the high energy covalent uridylyl-enzyme (UMP-enzyme) intermediate, which then reacts with Gal-1-P to produce UDP-Gal.

Catalytic Residues Roles

UniProt PDB* (1hxq)
His164 (main-C) His164A (main-C) Along with it's side chain, this residue is important for positioning and stabilising the catalytic His166. activator
Ser161 Ser161A Activates and stabilises the reaction intermediates. hydrogen bond donor, electrostatic stabiliser, steric role
His166 His166A Acts as the catalytic nucleophile. hydrogen bond donor, nucleophile, nucleofuge
His164 His164A Forms part of the zinc binding site. Also involved in maintaining the correct positioning of the substrates in the active site. hydrogen bond acceptor, metal ligand, steric role
Cys52, Cys55, His115 Cys52A, Cys55A, His115A Forms part of the zinc binding site. metal ligand
Asn153, Gln168, His166 (main-C) Asn153A, Gln168A, His166A (main-C) Holds the catalytically important water in position. These interactions help stabilise the phosphate intermediate. 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 nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, overall product formed, intermediate formation, enzyme-substrate complex cleavage, intermediate terminated, native state of enzyme regenerated

References

  1. Wedekind JE et al. (1996), Biochemistry, 35, 11560-11569. The Structure of Nucleotidylated Histidine-166 of Galactose-1-phosphate Uridylyltransferase Provides Insight into Phosphoryl Group Transfer†,‡. DOI:10.1021/bi9612677. PMID:8794735.
  2. McCorvie TJ et al. (2011), IUBMB Life, 63, 694-700. The structural and molecular biology of type I galactosemia: Enzymology of galactose 1-phosphate uridylyltransferase. DOI:10.1002/iub.511. PMID:21793161.
  3. Geeganage S et al. (2002), Methods Enzymol, 134-148. Galactose-1-Phosphate Uridylyltransferase: Kinetics of Formation and Reaction of Uridylyl-Enzyme Intermediate in Wild-Type and Specifically Mutated Uridylyltransferases. DOI:10.1016/s0076-6879(02)54010-6.
  4. Brenner C (2002), Biochemistry, 41, 9003-9014. Hint, Fhit, and GalT: function, structure, evolution, and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases. PMID:12119013.
  5. Geeganage S et al. (2000), Biochemistry, 39, 5397-5404. Roles of Two Conserved Amino Acid Residues in the Active Site of Galactose-1-Phosphate Uridylyltransferase:  An Essential Serine and a Nonessential Cysteine†. DOI:10.1021/bi992594s. PMID:10820011.
  6. Geeganage S et al. (1999), Biochemistry, 38, 13398-13406. Significance of Metal Ions in Galactose-1-Phosphate Uridylyltransferase:  An Essential Structural Zinc and a Nonessential Structural Iron†. DOI:10.1021/bi9910631. PMID:10529216.
  7. Thoden JB et al. (1997), Biochemistry, 36, 1212-1222. Structural Analysis of the H166G Site-Directed Mutant of Galactose-1-phosphate Uridylyltransferase Complexed with either UDP-glucose or UDP-galactose:  Detailed Description of the Nucleotide Sugar Binding Site†,‡. DOI:10.1021/bi9626517. PMID:9063869.
  8. Wedekind JE et al. (1995), Biochemistry, 34, 11049-11061. Three-dimensional structure of galactose-1-phosphate uridylyltransferase from Escherichia coli at 1.8 A resolution. PMID:7669762.

Step 1. His166 initiates a nucleophilic attack on the alpha-phosphate of the UDP-glucose substrate in a substitution reaction, eliminating glucose phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn153A hydrogen bond donor, activator
Ser161A hydrogen bond donor, electrostatic stabiliser, steric role
His164A metal ligand, steric role, hydrogen bond acceptor
His166A hydrogen bond donor
Gln168A electrostatic stabiliser, hydrogen bond donor
Cys52A metal ligand
Cys55A metal ligand
His115A metal ligand
His164A (main-C) activator
His166A (main-C) activator
His166A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, overall product formed, intermediate formation

Step 2. Galactose phosphate initiates a nucleophilic attack on the alpha-phosphate of the covalently bound UDP in a substitution reaction, eliminating His166.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn153A hydrogen bond donor, activator
Ser161A hydrogen bond donor, electrostatic stabiliser, steric role
His164A metal ligand, steric role, hydrogen bond acceptor
His166A hydrogen bond donor
Gln168A electrostatic stabiliser, hydrogen bond donor
Cys52A metal ligand
Cys55A metal ligand
His115A metal ligand
His166A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex cleavage, overall product formed, intermediate terminated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His166 initiates a nucleophilic attack on the alpha-phosphate of the UDP-glucose substrate in a substitution reaction, eliminating glucose phosphate.

Step 2. Galactose phosphate initiates a nucleophilic attack on the alpha-phosphate of the covalently bound UDP in a substitution reaction, eliminating His166.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, Anna Waters, Craig Porter