UDP-glucose 6-dehydrogenase

 

Bacterial UDP-glucose dehydrogenase is essential for the formation of the antiphagocytic capsule that protects many virulent bacteria from the host's immune system. The homodimeric enzyme is composed of an N-terminal NAD+ dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long central alpha helix.

The enzyme catalyses the NAD+ dependent oxidation of UDP-glucose to UDP-glucuronic acid. In mammals this is the substrate for UDP-glucuronosyl transferases in the liver. UDP-glucuronosyl transferases catalyse the formation of glucuronide conjugates with various substances e.g. bilirubin - aiding its excretion.

The active site contains residues contributed from the N- and C-terminal domains as well as from the central alpha-helix. Thr118 from the N-terminal forms a hydrogen bond to an ordered active site water molecule, that may be critical for the catalytic mechanism. Ser117 and Pro120 are also probably essential for proper orientation of the catalytic Thr118. The central alpha-helix contributes two active site residues; Lys204 and Asn208. Glu141 forms a hydrogen bond to the key catalytic residue Lys204. The C-terminal contributes two residues; Cys260 and Asp264, both having direct roles in the enzyme mechanism.

 

Reference Protein and Structure

Sequence
P0C0F4 UniProt (1.1.1.22) IPR028357 (Sequence Homologues) (PDB Homologues)
Biological species
Streptococcus pyogenes (Bacteria) Uniprot
PDB
1dli - THE FIRST STRUCTURE OF UDP-GLUCOSE DEHYDROGENASE (UDPGDH) REVEALS THE CATALYTIC RESIDUES NECESSARY FOR THE TWO-FOLD OXIDATION (2.31 Å) PDBe PDBsum 1dli
Catalytic CATH Domains
1.10.1040.10 CATHdb 3.40.50.720 CATHdb (see all for 1dli)
Click To Show Structure

Enzyme Reaction (EC:1.1.1.22)

water
CHEBI:15377ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
+
UDP-alpha-D-glucose(2-)
CHEBI:58885ChEBI
→
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
+
UDP-alpha-D-glucuronate(3-)
CHEBI:58052ChEBI
Alternative enzyme names: UDP-alpha-D-glucose:NAD oxidoreductase, UDP-D-glucose dehydrogenase, UDPG dehydrogenase, UDPG:NAD oxidoreductase, UDPglucose 6-dehydrogenase, UDPglucose dehydrogenase, UDPglucose:NAD 6-oxidoreductase, UDPglucose:NAD(+) oxidoreductase, Uridine diphosphate D-glucose dehydrogenase, Uridine diphosphate glucose dehydrogenase, Uridine diphosphoglucose dehydrogenase,

Enzyme Mechanism

Introduction

In this proposal, the primary C6'' hydroxyl of UDP-glucose is oxidised with the transfer of the pro-R hydride to C4 (NC4) on the si face of the nicotinamide ring of NAD+. This step is initiated by the transfer of a proton from the alcohol by water, activated by Asp264. Then, Cys260 initiates a nucleophilic attack on the newly formed carbonyl carbon. Collapse of the new oxyanion leads to the elimination of a second hydride ion. In the cleavage step, Glu145 deprotonates water, which attacks the carbonyl, carbon, eliminating cysteine.

Catalytic Residues Roles

UniProt PDB* (1dli)
Cys260 Cys260A Acts as a catalytic nucleophile. Thought to be negatively charged due to the optimum pH of the active site being around 9. covalently attached, nucleofuge, nucleophile
Asp264 Asp264A Acts as a general acid/base, activating the water molecule that performs the first proton abstraction of the mechanism. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu145 Glu145A Acts as a general acid/base, abstracting a proton from the water molecule that is responsible for the final hydrolysis step. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys204, Thr118, Asn208 Lys204A, Thr118A, Asn208A Act to stabilise the reactive intermediates. hydrogen bond donor, 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 elimination, hydride transfer, aromatic bimolecular nucleophilic addition, proton transfer, proton relay, bimolecular nucleophilic addition, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, overall reactant used, intermediate formation, enzyme-substrate complex cleavage, native state of enzyme regenerated, inferred reaction step

References

  1. Campbell RE et al. (2000), Biochemistry, 39, 7012-7023. The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation. DOI:10.2210/pdb1dlj/pdb. PMID:10841783.
  2. Chen YY et al. (2011), J Struct Biol, 175, 300-310. Conformational change upon product binding to Klebsiella pneumoniae UDP-glucose dehydrogenase: A possible inhibition mechanism for the key enzyme in polymyxin resistance. DOI:10.1016/j.jsb.2011.04.010. PMID:21536136.
  3. Rocha J et al. (2011), J Bacteriol, 193, 3978-3987. Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate. DOI:10.1128/jb.01076-10. PMID:21602353.
  4. Ge X et al. (2004), Eur J Biochem, 271, 14-22. Active site residues and mechanism of UDP-glucose dehydrogenase. PMID:14686915.

Step 1. Asp264 deprotonates water, which deprotonates the CH3OH group of UDP, which causes the elimination of a hydride which adds to NAD

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor
Asp264A hydrogen bond acceptor
Lys204A hydrogen bond donor
Asn208A hydrogen bond donor
Glu145A hydrogen bond acceptor
Asp264A proton acceptor

Chemical Components

ingold: bimolecular elimination, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, proton transfer, proton relay

Step 2. Cys260 initiates a nucleophilic attack on the formed carbonyl carbon in an addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Asn208A hydrogen bond donor, electrostatic stabiliser
Glu145A hydrogen bond acceptor
Cys260A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 3. The oxyanion collapses, eliminating a second hydride, which adds to a second molecule of NAD.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Glu145A hydrogen bond acceptor
Asn208A hydrogen bond donor, electrostatic stabiliser
Cys260A covalently attached

Chemical Components

ingold: unimolecular elimination by the conjugate base, hydride transfer, ingold: aromatic bimolecular nucleophilic addition

Step 4. Glu145 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor, activator
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Asn208A hydrogen bond donor, electrostatic stabiliser
Glu145A hydrogen bond acceptor
Cys260A covalently attached
Glu145A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 5. The oxyanion collapses, eliminating Cys260.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Thr118A hydrogen bond acceptor, electrostatic stabiliser
Asn208A hydrogen bond donor, electrostatic stabiliser
Cys260A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage

Step 6. In an inferred return step, two water molecules deprotonate Glu145 and Asp264.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp264A hydrogen bond donor
Thr118A hydrogen bond acceptor
Glu145A hydrogen bond donor
Asp264A proton donor
Glu145A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Asp264 deprotonates water, which deprotonates the CH3OH group of UDP, which causes the elimination of a hydride which adds to NAD

Step 2. Cys260 initiates a nucleophilic attack on the formed carbonyl carbon in an addition reaction.

Step 3. The oxyanion collapses, eliminating a second hydride, which adds to a second molecule of NAD.

Step 4. Glu145 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate.

Step 5. The oxyanion collapses, eliminating Cys260.

Step 6. In an inferred return step, two water molecules deprotonate Glu145 and Asp264.

Products.

Introduction

In this proposal, the primary C6'' hydroxyl of UDP-glucose is oxidised with the transfer of the pro-R hydride to C4 (NC4) on the si face of the nicotinamide ring of NAD+. This step is initiated by the transfer of a proton from the alcohol by Lys204. Then, Cys260 initiates a nucleophilic attack on the newly formed carbonyl carbon. Collapse of the new oxyanion leads to the elimination of a second hydride ion. In the cleavage step, Glu145 deprotonates water, which attacks the carbonyl, carbon, eliminating cysteine.

Catalytic Residues Roles

UniProt PDB* (1dli)
Lys204 Lys204A Acts as a general acid/base, responsible for the initial proton abstraction. proton acceptor, hydrogen bond donor, electrostatic stabiliser, proton donor
Cys260 Cys260A Acts as a catalytic nucleophile. Thought to be negatively charged due to the optimum pH of the active site being around 9 covalently attached, nucleofuge, nucleophile
Asp264 Asp264A Activates the catalytic water through hydrogen bonding. hydrogen bond acceptor, hydrogen bond donor
Glu145 Glu145A Acts as a general acid/base, abstracting a proton from the water molecule that is responsible for the final hydrolysis step. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Thr118, Asn208 Thr118A, Asn208A Act to stabilise the reactive intermediates. activator, hydrogen bond acceptor, 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 elimination, hydride transfer, aromatic bimolecular nucleophilic addition, proton transfer, overall product formed, bimolecular nucleophilic addition, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, overall reactant used, intermediate formation, enzyme-substrate complex cleavage, native state of enzyme regenerated, inferred reaction step

References

  1. Campbell RE et al. (2000), Biochemistry, 39, 7012-7023. The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation. DOI:10.2210/pdb1dlj/pdb. PMID:10841783.
  2. Chen YY et al. (2011), J Struct Biol, 175, 300-310. Conformational change upon product binding to Klebsiella pneumoniae UDP-glucose dehydrogenase: A possible inhibition mechanism for the key enzyme in polymyxin resistance. DOI:10.1016/j.jsb.2011.04.010. PMID:21536136.
  3. Ge X et al. (2004), Eur J Biochem, 271, 14-22. Active site residues and mechanism of UDP-glucose dehydrogenase. PMID:14686915.

Step 1. Lys204 deprotonates the CH3OH group of UDP, which causes the elimination of a hydride which adds to NAD.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor
Asp264A hydrogen bond acceptor
Lys204A hydrogen bond donor
Asn208A hydrogen bond donor
Glu145A hydrogen bond acceptor
Lys204A proton acceptor

Chemical Components

ingold: bimolecular elimination, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, proton transfer, overall product formed

Step 2. Cys260 initiates a nucleophilic attack on the formed carbonyl carbon in an addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Asn208A hydrogen bond donor, electrostatic stabiliser
Glu145A hydrogen bond acceptor
Cys260A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 3. The oxyanion collapses, eliminating a second hydride, which adds to a second molecule of NAD.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Glu145A hydrogen bond acceptor
Asn208A hydrogen bond donor, electrostatic stabiliser
Cys260A covalently attached

Chemical Components

ingold: unimolecular elimination by the conjugate base, hydride transfer, ingold: aromatic bimolecular nucleophilic addition

Step 4. Glu145 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr118A hydrogen bond acceptor, activator
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Asn208A hydrogen bond donor, electrostatic stabiliser
Glu145A hydrogen bond acceptor
Cys260A covalently attached
Glu145A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 5. The oxyanion collapses, eliminating Cys260.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp264A hydrogen bond donor
Lys204A hydrogen bond donor, electrostatic stabiliser
Thr118A hydrogen bond acceptor, electrostatic stabiliser
Asn208A hydrogen bond donor, electrostatic stabiliser
Cys260A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage

Step 6. In an inferred return step, two water molecules deprotonate Glu145 and Asp264.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp264A hydrogen bond donor
Thr118A hydrogen bond acceptor
Glu145A hydrogen bond donor, proton donor
Lys204A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Lys204 deprotonates the CH3OH group of UDP, which causes the elimination of a hydride which adds to NAD.

Step 2. Cys260 initiates a nucleophilic attack on the formed carbonyl carbon in an addition reaction.

Step 3. The oxyanion collapses, eliminating a second hydride, which adds to a second molecule of NAD.

Step 4. Glu145 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound substrate.

Step 5. The oxyanion collapses, eliminating Cys260.

Step 6. In an inferred return step, two water molecules deprotonate Glu145 and Asp264.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, James W. Murray, Craig Porter