3-dehydro-L-gulonate-6-phosphate decarboxylase

 

3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), isolated from Escherichia coli, catalyses the decarboxylation of 3-keto-L-gulonate 6-phosphate to L-xylulose 5-phosphate and carbon dioxide. KGPDB is a member of the orotidine 5'-monophosphate decarboxylase suprafamily, in that it shares a common active site architecture but not substrate specificity or mechanism. KGPDC is promiscuous and can also catalyse a low level of the D-arabino-hex-3-ulose 6-phosphate synthase (HPS) reaction: D-ribulose 5-phosphate and formaldehyde to D-arabino-hex-3-ulose 6-phosphate.

KGPDC exists as a homodimer with two active sites. Residues from both subunits can be found in each active site.

 

Reference Protein and Structure

Sequence
P39304 UniProt (4.1.1.85) IPR023942 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1q6l - Structure of 3-keto-L-gulonate 6-phosphate decarboxylase with bound L-threonohydroxamate 4-phosphate (1.8 Å) PDBe PDBsum 1q6l
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1q6l)
Cofactors
Water (2), Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.1.1.85)

3-dehydro-L-gulonate 6-phosphate
CHEBI:58774ChEBI
+
hydron
CHEBI:15378ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
L-xylulose 5-phosphate(2-)
CHEBI:57829ChEBI
Alternative enzyme names: 3-keto-L-gulonate 6-phosphate decarboxylase, UlaD, SgaH, SgbH, KGPDC, 3-dehydro-L-gulonate-6-phosphate carboxy-lyase,

Enzyme Mechanism

Introduction

Si-Face mechanism - A hydrophobic pocket surrounding the carboxylate of 3-keto-L-gulonate 6-phosphate destabilises the ground state and favours the decarboxylation to form a 1,2-cis-enediolate intermediate. This intermediate is stabilised by Mg(II) coordinating to the negatively-charged C2 oxygen, by Lys64 forming hydrogen bonds to the C1 and C2 oxygens, and by Asp67 forming a hydrogen bond to the C1 oxygen. The carbonyl reforms and the C1 position is protonated by either a si-face water or a re-face water to form L-xylulose 5-phosphate. His136 protonates the si-face water, allowing it to act as a proton shuttle. Arg139 protonates the re-face water so that it too can act as a proton shuttle. Protonation by the si-face water is favoured in a roughly 2:1 ratio.

Catalytic Residues Roles

UniProt PDB* (1q6l)
Arg139 Arg139A Arg139 can protonate the re-face water, allowing the water to act as a proton shuttle to the re-face of the enediolate intermediate. hydrogen bond donor
Asp67 Asp67B Asp67 forms a hydrogen bond to the C1 oxygen atom of the intermediate. This stabilises both the intermediate and the transition state for the formation of the intermediate. hydrogen bond acceptor, electrostatic stabiliser
His136 His136A His136 can protonate the si-face water, allowing the water to act as a proton shuttle to the si-face of the enediolate intermediate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys64 Lys64A Lys64 forms hydrogen bonds to the C1 and C2 oxygen atoms of the intermediate. This stabilises both the intermediate and the transition state for the formation of the intermediate. attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser
Glu112 Glu112A Glu112 forms a hydrogen bond to N-delta of His136. This is thought to stabilise the protonated state of His136, aiding in general acid catalysis. increase basicity, hydrogen bond acceptor, electrostatic stabiliser, increase acidity
Ile37, Leu72, Ala68, Thr36 Ile37A, Leu72B, Ala68B, Thr36A Forms part of a hydrophobic pocket that may destabilise the ground state by surrounding the negatively-charged carboxylate of the substrate. ground state destabiliser
*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

unimolecular elimination by the conjugate base, decarboxylation, intermediate formation, overall product formed, overall reactant used, assisted keto-enol tautomerisation, proton transfer, intermediate terminated, proton relay, native state of enzyme regenerated, inferred reaction step

References

  1. Wise EL et al. (2003), Biochemistry, 42, 12133-12142. Structural Evidence for a 1,2-Enediolate Intermediate in the Reaction Catalyzed by 3-Keto-l-Gulonate 6-Phosphate Decarboxylase, a Member of the Orotidine 5‘-Monophosphate Decarboxylase Suprafamily†,‡. DOI:10.1021/bi0348819. PMID:14567674.
  2. Li T et al. (2012), Bioorg Chem, 43, 2-14. Decarboxylation mechanisms in biological system. DOI:10.1016/j.bioorg.2012.03.001. PMID:22534166.
  3. Yew WS et al. (2004), Biochemistry, 43, 6427-6437. Evolution of Enzymatic Activities in the Orotidine 5‘-Monophosphate Decarboxylase Suprafamily:  Mechanistic Evidence for a Proton Relay System in the Active Site of 3-Keto-l-gulonate 6-Phosphate Decarboxylase†. DOI:10.1021/bi049741t. PMID:15157077.
  4. Wise EL et al. (2004), Biochemistry, 43, 6438-6446. Evolution of Enzymatic Activities in the Orotidine 5‘-Monophosphate Decarboxylase Suprafamily:  Crystallographic Evidence for a Proton Relay System in the Active Site of 3-Keto-l-gulonate 6-Phosphate Decarboxylase†,‡. DOI:10.1021/bi0497392. PMID:15157078.

Step 1. The substrate decarboxylates to form the 1,2-cis-enediolate intermediate and carbon dioxide. It has been suggested that the ground state is destabilised due to the interactions between the hydrophobic pocket and the negatively-charged carboxylate of 3-dehydro-L-gulonate 6-phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg139A hydrogen bond donor
Lys64A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Glu112A hydrogen bond acceptor
His136A hydrogen bond donor
Asp67B hydrogen bond acceptor, electrostatic stabiliser
Thr36A ground state destabiliser
Ile37A ground state destabiliser
Ala68B ground state destabiliser
Leu72B ground state destabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, decarboxylation, intermediate formation, overall product formed, overall reactant used

Step 2. The enediolate can be protonated from either the re- or si-face. The si-face water is protonated by His136 to allow it to act as a proton shuttle. However, if the protonation occurs at the re-face, Arg139A has been proposed to be the proton source. Since protonation at the si-face is favoured in a roughly 2:1 ratio, this is the mechanism that has been shown.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp67B hydrogen bond acceptor, electrostatic stabiliser
Glu112A hydrogen bond acceptor, increase acidity, electrostatic stabiliser
His136A hydrogen bond donor
Lys64A hydrogen bond donor, electrostatic stabiliser
Arg139A hydrogen bond donor
His136A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, intermediate terminated, overall product formed, proton relay

Step 3. An acid, assumed to be water, protonates His136.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His136A hydrogen bond donor, hydrogen bond acceptor
Glu112A increase basicity, hydrogen bond acceptor
Arg139A hydrogen bond donor
His136A proton acceptor

Chemical Components

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

Step 1. The substrate decarboxylates to form the 1,2-cis-enediolate intermediate and carbon dioxide. It has been suggested that the ground state is destabilised due to the interactions between the hydrophobic pocket and the negatively-charged carboxylate of 3-dehydro-L-gulonate 6-phosphate.

Step 2. The enediolate can be protonated from either the re- or si-face. The si-face water is protonated by His136 to allow it to act as a proton shuttle. However, if the protonation occurs at the re-face, Arg139A has been proposed to be the proton source. Since protonation at the si-face is favoured in a roughly 2:1 ratio, this is the mechanism that has been shown.

Step 3. An acid, assumed to be water, protonates His136.

Products.

Introduction

Re-Face mechanism - A hydrophobic pocket surrounding the carboxylate of 3-keto-L-gulonate 6-phosphate destabilises the ground state and favours the decarboxylation to form a 1,2-cis-enediolate intermediate. This intermediate is stabilised by Mg(II) coordinating to the negatively-charged C2 oxygen, by Lys64 forming hydrogen bonds to the C1 and C2 oxygens, and by Asp67 forming a hydrogen bond to the C1 oxygen. The carbonyl reforms and the C1 position is protonated by either a si-face water or a re-face water to form L-xylulose 5-phosphate amd in this mechanism we show the protonation by the re-face water which is protonated by Arg139. This is the least favoured mechanism as si-face protonation is favoured 2:1 ratio.

Catalytic Residues Roles

UniProt PDB* (1q6l)
Arg139 Arg139A Arg139 can protonate the re-face water, allowing the water to act as a proton shuttle to the re-face of the enediolate intermediate. hydrogen bond donor, proton acceptor, proton donor
Asp67 Asp67B Asp67 forms a hydrogen bond to the C1 oxygen atom of the intermediate. This stabilises both the intermediate and the transition state for the formation of the intermediate. hydrogen bond acceptor, electrostatic stabiliser
His136 His136A His136 can protonate the si-face water, allowing the water to act as a proton shuttle to the si-face of the enediolate intermediate. hydrogen bond acceptor, hydrogen bond donor
Lys64 Lys64A Lys64 forms hydrogen bonds to the C1 and C2 oxygen atoms of the intermediate. This stabilises both the intermediate and the transition state for the formation of the intermediate. attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser
Glu112 Glu112A Glu112 forms a hydrogen bond to N-delta of His136. This is thought to stabilise the protonated state of His136, aiding in general acid catalysis. increase basicity, hydrogen bond acceptor, electrostatic stabiliser, increase acidity
Ile37, Leu72, Ala68, Thr36 Ile37A, Leu72B, Ala68B, Thr36A Forms part of a hydrophobic pocket that may destabilise the ground state by surrounding the negatively-charged carboxylate of the substrate. ground state destabiliser
*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

unimolecular elimination by the conjugate base, decarboxylation, intermediate formation, overall product formed, overall reactant used, assisted keto-enol tautomerisation, proton transfer, intermediate terminated, proton relay, native state of enzyme regenerated, inferred reaction step

References

  1. Yew WS et al. (2004), Biochemistry, 43, 6427-6437. Evolution of Enzymatic Activities in the Orotidine 5‘-Monophosphate Decarboxylase Suprafamily:  Mechanistic Evidence for a Proton Relay System in the Active Site of 3-Keto-l-gulonate 6-Phosphate Decarboxylase†. DOI:10.1021/bi049741t. PMID:15157077.
  2. Wise EL et al. (2004), Biochemistry, 43, 6438-6446. Evolution of Enzymatic Activities in the Orotidine 5‘-Monophosphate Decarboxylase Suprafamily:  Crystallographic Evidence for a Proton Relay System in the Active Site of 3-Keto-l-gulonate 6-Phosphate Decarboxylase†,‡. DOI:10.1021/bi0497392. PMID:15157078.

Step 1. The substrate decarboxylates to form the 1,2-cis-enediolate intermediate and carbon dioxide. It has been suggested that the ground state is destabilised due to the interactions between the hydrophobic pocket and the negatively-charged carboxylate of 3-dehydro-L-gulonate 6-phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg139A hydrogen bond donor
Leu72B ground state destabiliser
Ala68B ground state destabiliser
Ile37A ground state destabiliser
Thr36A ground state destabiliser
Lys64A attractive charge-charge interaction, electrostatic stabiliser, hydrogen bond donor
Glu112A hydrogen bond acceptor
His136A hydrogen bond donor
Asp67B hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, decarboxylation, intermediate formation, overall product formed, overall reactant used

Step 2. The enediolate can be protonated from either the re- or si-face. The si-face water is protonated by His136 to allow it to act as a proton shuttle. However, if the protonation occurs at the re-face, Arg139A has been proposed to be the proton source. Since protonation at the si-face is favoured in a roughly 2:1 ratio, this is the mechanism that has been shown.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg139A hydrogen bond donor
Asp67B hydrogen bond acceptor, electrostatic stabiliser
Glu112A hydrogen bond acceptor, increase acidity, electrostatic stabiliser
His136A hydrogen bond donor
Lys64A hydrogen bond donor, electrostatic stabiliser
Arg139A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, intermediate terminated, overall product formed, proton relay

Step 3. An acid, assumed to be water, protonates His136.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg139A hydrogen bond donor
His136A hydrogen bond donor, hydrogen bond acceptor
Glu112A increase basicity, hydrogen bond acceptor
Arg139A proton acceptor

Chemical Components

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

Step 1. The substrate decarboxylates to form the 1,2-cis-enediolate intermediate and carbon dioxide. It has been suggested that the ground state is destabilised due to the interactions between the hydrophobic pocket and the negatively-charged carboxylate of 3-dehydro-L-gulonate 6-phosphate.

Step 2. The enediolate can be protonated from either the re- or si-face. The si-face water is protonated by His136 to allow it to act as a proton shuttle. However, if the protonation occurs at the re-face, Arg139A has been proposed to be the proton source. Since protonation at the si-face is favoured in a roughly 2:1 ratio, this is the mechanism that has been shown.

Step 3. An acid, assumed to be water, protonates His136.

Products.

Contributors

Judith A. Reeks, Gemma L. Holliday, Charity Hornby