Ribulose-phosphate 3-epimerase (cytoplasmic)

 

Ribulose-phosphate 3-epimerase (EC:5.1.3.1) (also known as RPE, pentose-5-phosphate 3-epimerase or PPE) is the enzyme that converts D-ribulose 5-phosphate (Ru5P) into D-xylulose 5-phosphate in Calvin's reductive pentose phosphate cycle. RPE has been found in a wide range of bacteria, archaebacteria, fungi and plants. All the proteins have from 209 to 241 amino acid residues and have a TIM barrel structure.

This enzyme participates in both the oxidative and reductive pentose phosphate pathways and is thus an amphibolic enzyme [PMID:12547196]. There is some discussion as to whether this enzyme is zinc dependent or not. No zinc dependence has been reported [PMID:12547196]. The zinc independent (in which a water molecule is bound in place of a zinc ion to the two histidine residues that are seen as the zinc binding ligands as well as the two catalytic aspartate residues) mechanism has issues relating to how the intermediate formed is stabilised, and it has been suggested that the three strictly conserved methionines (Met40, Met71 and Met144) act as a transient 'electrostatic cushion' [PMID:10191144]. However, the alternative in which the zinc ion stabilises the oxyanion formed is more attractive [PMID:12547196] with the methionine residues aiding in this process and ensuring a hydrophobic and thus proton free environment. The actual mechanisms (with respect to the bonds formed and cleaved) are identical between the two proposals, the only difference lies in how the intermediate is stabilised [PMID:10191144, PMID:12547196, PMID:15333955].

 

Reference Protein and Structure

Sequence
Q9SE42 UniProt (5.1.3.1) IPR000056 (Sequence Homologues) (PDB Homologues)
Biological species
Oryza sativa Japonica Group (Japanese rice) Uniprot
PDB
1h1z - The structure of the cytosolic D-ribulose-5-phosphate 3-epimerase from rice complexed with sulfate and zinc (3.4 Å) PDBe PDBsum 1h1z
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1h1z)
Cofactors
Zinc(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:5.1.3.1)

D-ribulose 5-phosphate(2-)
CHEBI:58121ChEBI
→
D-xylulose 5-phosphate(2-)
CHEBI:57737ChEBI
Alternative enzyme names: D-ribulose 5-phosphate epimerase, D-ribulose phosphate-3-epimerase, D-ribulose-5-P 3-epimerase, D-xylulose-5-phosphate 3-epimerase, Erythrose-4-phosphate isomerase, Pentose-5-phosphate 3-epimerase, Phosphoketopentose 3-epimerase, Phosphoketopentose epimerase, Phosphoribulose epimerase, Ribulose 5-phosphate 3-epimerase, Xylulose phosphate 3-epimerase, D-ribulose-5-phosphate epimerase, Erythrose-4-phosphate epimerase,

Enzyme Mechanism

Introduction

Asp38 deprotonates the C3 of the substrate molecule, causing a rearrangement of the double bonds and the formation of the enolate form. The enolate then collapses back to the keto form with concomitant deprotonation of Asp178, forming the xylulose product.

Catalytic Residues Roles

UniProt PDB* (1h1z)
Asp179 Asp178(179)A Forms part of the zinc binding site. Also acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor
Asp39 Asp38(39)A Forms part of the zinc binding site and acts as a general acid/base. Upon protonation, it leaves the Zn(II) ligand sphere leaving room for the negatively charged intermediate to be stabilised as a zinc ligand [PMID:12547196]. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor
Ser12 Ser11(12)A Activates and stabilises Asp38. hydrogen bond donor, electrostatic stabiliser, increase acidity
Met145, Met72, Met41 Met144(145)A, Met71(72)A, Met40(41)A These methionine residues aid catalysis by ensuring a hydrophobic and thus proton free environment. electrostatic stabiliser
His37, His70 His36(37)A, His69(70)A Forms part of the divalent metal binding site. metal ligand
*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

proton transfer, assisted keto-enol tautomerisation, intermediate formation, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Jelakovic S et al. (2003), J Mol Biol, 326, 127-135. Structure and Catalytic Mechanism of the Cytosolic d-Ribulose-5-phosphate 3-Epimerase from Rice. DOI:10.1016/s0022-2836(02)01374-8. PMID:12547196.
  2. Liang W et al. (2011), FASEB J, 25, 497-504. Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate: new insights from structural and biochemical studies on human RPE. DOI:10.1096/fj.10-171207. PMID:20923965.
  3. Akana J et al. (2006), Biochemistry, 45, 2493-2503. D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily. DOI:10.1021/bi052474m. PMID:16489742.
  4. Wise EL et al. (2004), Acta Crystallogr D Biol Crystallogr, 60, 1687-1690. Structure ofD-ribulose 5-phosphate 3-epimerase fromSynechocystisto 1.6 Å resolution. DOI:10.1107/s0907444904015896. PMID:15333955.
  5. Kopp J et al. (1999), J Mol Biol, 287, 761-771. Structure and mechanism of the amphibolic enzyme d-ribulose-5-phosphate 3-epimerase from potato chloroplasts. DOI:10.1006/jmbi.1999.2643. PMID:10191144.

Step 1. Asp38 deprotonates the C3 of the substrate molecule, causing a rearrangement of the double bonds and the formation of the enolate form. Upon protonation, Asp38 leaves the Zn(II) ligand sphere leaving room for the negatively charged intermediate to be stabilised as a zinc ligand [PMID:12547196]. Three conserved methionines are essential for maintaining a proton free environment to ensure that the epimerisation (and not isomerisation) occurs [PMID:12547196].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp178(179)A hydrogen bond donor
Asp38(39)A hydrogen bond acceptor
Ser11(12)A hydrogen bond donor, electrostatic stabiliser
Asp178(179)A metal ligand
Asp38(39)A metal ligand
His36(37)A metal ligand
His69(70)A metal ligand
Met40(41)A electrostatic stabiliser
Met71(72)A electrostatic stabiliser
Met144(145)A electrostatic stabiliser
Asp38(39)A proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, intermediate formation, overall reactant used

Step 2. The enolate collapses back to the keto form with concomitant deprotonation of Asp178, forming the xylulose product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp178(179)A hydrogen bond donor
Asp38(39)A hydrogen bond acceptor
Ser11(12)A hydrogen bond donor, electrostatic stabiliser
Asp178(179)A metal ligand
His36(37)A metal ligand
His69(70)A metal ligand
Met40(41)A electrostatic stabiliser
Met71(72)A electrostatic stabiliser
Met144(145)A electrostatic stabiliser
Asp178(179)A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, overall product formed

Step 3. Once the product has left the active site the two catalytic aspartates are open to bulk solvent and it is expected that the return to the correct protonation states is thus from bulk solvent [PMID:10191144, PMID:12547196]. This step is only necessary if the enzyme is to perform the reaction again in the same direction as described here. The enzyme catalyses the epimerisation in both directions, however.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp178(179)A hydrogen bond acceptor
Asp38(39)A hydrogen bond acceptor, hydrogen bond donor
Ser11(12)A hydrogen bond donor, increase acidity, electrostatic stabiliser
Asp178(179)A metal ligand
Asp38(39)A metal ligand
His36(37)A metal ligand
His69(70)A metal ligand
Asp38(39)A proton donor
Asp178(179)A proton acceptor

Chemical Components

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

Step 1. Asp38 deprotonates the C3 of the substrate molecule, causing a rearrangement of the double bonds and the formation of the enolate form. Upon protonation, Asp38 leaves the Zn(II) ligand sphere leaving room for the negatively charged intermediate to be stabilised as a zinc ligand [PMID:12547196]. Three conserved methionines are essential for maintaining a proton free environment to ensure that the epimerisation (and not isomerisation) occurs [PMID:12547196].

Step 2. The enolate collapses back to the keto form with concomitant deprotonation of Asp178, forming the xylulose product.

Step 3. Once the product has left the active site the two catalytic aspartates are open to bulk solvent and it is expected that the return to the correct protonation states is thus from bulk solvent [PMID:10191144, PMID:12547196]. This step is only necessary if the enzyme is to perform the reaction again in the same direction as described here. The enzyme catalyses the epimerisation in both directions, however.

Products.

Introduction

The zinc-independent proposal in which the conserved methionines are responsible for the stabilisation of the reactive intermediates and transition states. The mechanism itself is identical to the primary proposal in which Asp38 deprotonates the C3 of the substrate molecule, causing a rearrangement of the double bonds and the formation of the enolate form. The enolate then collapses back to the keto form with concomitant deprotonation of Asp178, forming the xylulose product.

Catalytic Residues Roles

UniProt PDB* (1h1z)
Asp179, Asp39 Asp178(179)A, Asp38(39)A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Ser12 Ser11(12)A Activates and stabilises Asp38. hydrogen bond donor, electrostatic stabiliser, increase acidity
Met145, Met72, Met41 Met144(145)A, Met71(72)A, Met40(41)A These three methionine residues are highly conserved and thought to act as a transient 'electrostatic cushion' [PMID:10191144] that stabilises the reactive intermediates and transition states formed during the course of the reaction. These methionine residues also aid catalysis by ensuring a hydrophobic and thus proton free environment. 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

proton transfer, assisted keto-enol tautomerisation, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Kopp J et al. (1999), J Mol Biol, 287, 761-771. Structure and mechanism of the amphibolic enzyme d-ribulose-5-phosphate 3-epimerase from potato chloroplasts. DOI:10.1006/jmbi.1999.2643. PMID:10191144.
  2. Jelakovic S et al. (2003), J Mol Biol, 326, 127-135. Structure and Catalytic Mechanism of the Cytosolic d-Ribulose-5-phosphate 3-Epimerase from Rice. DOI:10.1016/s0022-2836(02)01374-8. PMID:12547196.

Step 1. Asp38 deprotonates the C3 of the substrate molecule, causing a rearrangement of the double bonds and the formation of the enolate form. Three conserved methionines are essential for maintaining a proton free environment to ensure that the epimerisation (and not isomerisation) occurs [PMID:12547196].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp178(179)A hydrogen bond donor
Asp38(39)A hydrogen bond acceptor
Ser11(12)A hydrogen bond donor, electrostatic stabiliser
Met40(41)A electrostatic stabiliser
Met71(72)A electrostatic stabiliser
Met144(145)A electrostatic stabiliser
Asp38(39)A proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, overall reactant used

Step 2. The enolate collapses back to the keto form with concomitant deprotonation of Asp178, forming the xylulose product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser11(12)A electrostatic stabiliser
Met40(41)A electrostatic stabiliser
Met71(72)A electrostatic stabiliser
Met144(145)A electrostatic stabiliser
Asp178(179)A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, overall product formed

Step 3. Once the product has left the active site the two catalytic aspartates are open to bulk solvent and it is expected that the return to the correct protonation states is thus from bulk solvent [PMID:10191144, PMID:12547196]. This step is only necessary if the enzyme is to perform the reaction again in the same direction as described here. The enzyme catalyses the epimerisation in both directions, however.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp178(179)A hydrogen bond acceptor
Asp38(39)A hydrogen bond acceptor, hydrogen bond donor
Ser11(12)A hydrogen bond donor, increase acidity, electrostatic stabiliser
Asp38(39)A proton donor
Asp178(179)A proton acceptor

Chemical Components

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

Step 1. Asp38 deprotonates the C3 of the substrate molecule, causing a rearrangement of the double bonds and the formation of the enolate form. Three conserved methionines are essential for maintaining a proton free environment to ensure that the epimerisation (and not isomerisation) occurs [PMID:12547196].

Step 2. The enolate collapses back to the keto form with concomitant deprotonation of Asp178, forming the xylulose product.

Step 3. Once the product has left the active site the two catalytic aspartates are open to bulk solvent and it is expected that the return to the correct protonation states is thus from bulk solvent [PMID:10191144, PMID:12547196]. This step is only necessary if the enzyme is to perform the reaction again in the same direction as described here. The enzyme catalyses the epimerisation in both directions, however.

Products.

Contributors

Gemma L. Holliday, Christian Drew, Craig Porter