DTDP-6-deoxy-D-xylo-4-hexulose 3,5 epimerase

 

dTDP-4-dehydrorhamnose 3,5-epimerase is the third enzyme in the biosynthetic pathway of dTDP-L-rhamnose, a saccharide required for the virulence of some pathogenic bacteria. The pathway does not exist in humans and therefore the enzymes in it are potential drug targets. The enzyme fold is unlike that of any other known epimerase and so represents a novel class of epimerase.

 

Reference Protein and Structure

Sequence
Q9HU21 UniProt (5.1.3.13) IPR000888 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas aeruginosa PAO1 (Bacteria) Uniprot
PDB
2ixj - RmlC P aeruginosa native (2.54 Å) PDBe PDBsum 2ixj
Catalytic CATH Domains
2.60.120.10 CATHdb (see all for 2ixj)
Click To Show Structure

Enzyme Reaction (EC:5.1.3.13)

dTDP-4-dehydro-6-deoxy-alpha-D-glucose(2-)
CHEBI:57649ChEBI
→
dTDP-4-dehydro-beta-L-rhamnose(2-)
CHEBI:62830ChEBI
Alternative enzyme names: TDP-4-keto-L-rhamnose-3,5-epimerase, TDP-4-ketorhamnose 3,5-epimerase, dTDP-L-rhamnose synthetase, Thymidine diphospho-4-ketorhamnose 3,5-epimerase, dTDP-4-keto-6-deoxyglucose 3,5-epimerase, dTDP-4-dehydro-6-deoxy-D-glucose 3,5-epimerase,

Enzyme Mechanism

Introduction

The reaction is thought to be a novel one. His65 abstracts a proton from the acidic 5' position, accelerated by the electrostatic interaction with Glu171, to form the enolate-tautomer. Inorder for His65 to act as a general base in the following reaction steps the residue must be deprotonated. Tyr134 acts as a general base towards the C3' carbon in a stereo-selective manner. The second epimerisation is initiated by His65 at the C3' position. The enolate collapses with stereo-selective reprotonation of the C3' by Tyr134. The regeneration of the active site by reprotonation of Tyr134 and His65.

Catalytic Residues Roles

UniProt PDB* (2ixj)
His62 His65A Acts as a general acid/base; abstracts a proton from the acidic 5' position. proton acceptor, proton donor
Lys71 Lys74A Stabilises the negatively charged intermediates and transition states formed during the course of the reaction. electrostatic stabiliser
Tyr131 Tyr134A Acts as a general acid/base towards the C3' carbon in a stereo-selective manner. proton relay, proton acceptor, proton donor
Asp168 Asp171A Activates the catalytic histidine residue. increase basicity, electrostatic stabiliser, increase acidity
*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, inferred reaction step, overall product formed, intermediate collapse, native state of enzyme regenerated

References

  1. Dong C et al. (2007), J Mol Biol, 365, 146-159. RmlC, a C3′ and C5′ Carbohydrate Epimerase, Appears to Operate via an Intermediate with an Unusual Twist Boat Conformation. DOI:10.1016/j.jmb.2006.09.063. PMID:17046787.
  2. Tello M et al. (2008), Chembiochem, 9, 1295-1302. Tyl1a, a TDP-6-deoxy-D-xylo-4-hexulose 3,4-isomerase from Streptomyces fradiae: structure prediction, mutagenesis and solvent isotope incorporation experiments to investigate reaction mechanism. DOI:10.1002/cbic.200800021. PMID:18425854.
  3. Giraud MF et al. (2000), Nat Struct Biol, 7, 398-402. RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase. DOI:10.1038/75178. PMID:10802738.

Step 1. His65 abstracts a proton from the acidic 5' position, accelerated by the electrostatic interaction with Asp171, to form the enolate-tautomer. Biochemical investigations suggest that epimerisation at the 5' centre occurs before that at the 3' centre [PMID:17046787].

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Catalytic Residues Roles

Residue Roles
Lys74A electrostatic stabiliser
Asp171A electrostatic stabiliser, increase basicity
His65A proton acceptor

Chemical Components

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

Step 2. Inorder for His65 to act as a general base in the following reaction steps the residue must be deprotonated. Crystallography shows solvent molecules to be present within the active site [PMID:17046787]. Water may potentially act as a specific base, deprotonating His65 for the next round of catalysis.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys74A electrostatic stabiliser
Asp171A electrostatic stabiliser, increase acidity
His65A proton donor

Chemical Components

proton transfer, inferred reaction step

Step 3. Tyr134 acts as a general base towards the C3' carbon in a stereo-selective manner. Solvent molecules within the active site have been shown to be involved in the stereo-selectve reprotonation stages, although mutagenesis and kinetics studies have shown Tyr134 to be crucial for catalyic activity [PMID:17046787].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys74A electrostatic stabiliser
Asp171A electrostatic stabiliser
Tyr134A proton acceptor, proton relay, proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, intermediate formation

Step 4. The second epimerisation is initiated by His65 at the C3' position. Inorder for the C3' proton to be made acidic enough to be removed, it must be orientated orthogonally to the carbonyl group. This is acheived by a ring inversion at the C4' carbonyl between step four and step five [PMID:17046787].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys74A electrostatic stabiliser
Asp171A electrostatic stabiliser, increase basicity
His65A proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, intermediate formation

Step 5. The enolate collapses with stereo-selective reprotonation of the C3' by Tyr134.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys74A electrostatic stabiliser
Asp171A electrostatic stabiliser
Tyr134A proton donor

Chemical Components

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

Step 6. The regeneration of the active site by reprotonation of Tyr134 and His65.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp171A increase acidity
His65A proton donor
Tyr134A proton acceptor

Chemical Components

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

Step 1. His65 abstracts a proton from the acidic 5' position, accelerated by the electrostatic interaction with Asp171, to form the enolate-tautomer. Biochemical investigations suggest that epimerisation at the 5' centre occurs before that at the 3' centre [PMID:17046787].

Step 2. Inorder for His65 to act as a general base in the following reaction steps the residue must be deprotonated. Crystallography shows solvent molecules to be present within the active site [PMID:17046787]. Water may potentially act as a specific base, deprotonating His65 for the next round of catalysis.

Step 3. Tyr134 acts as a general base towards the C3' carbon in a stereo-selective manner. Solvent molecules within the active site have been shown to be involved in the stereo-selectve reprotonation stages, although mutagenesis and kinetics studies have shown Tyr134 to be crucial for catalyic activity [PMID:17046787].

Step 4. The second epimerisation is initiated by His65 at the C3' position. Inorder for the C3' proton to be made acidic enough to be removed, it must be orientated orthogonally to the carbonyl group. This is acheived by a ring inversion at the C4' carbonyl between step four and step five [PMID:17046787].

Step 5. The enolate collapses with stereo-selective reprotonation of the C3' by Tyr134.

Step 6. The regeneration of the active site by reprotonation of Tyr134 and His65.

Products.

Contributors

Sophie T. Williams, James W. Murray, Craig Porter, Gemma L. Holliday, James Willey