1-deoxy-D-xylulose 5-phosphate synthase

 

1-Deoxy-D-xylulose-5-phosphate synthase (DXS ) is a regulatory enzyme of the mevalonate-independent pathway involved in terpenoid biosynthesis. DXP synthase is a thiamine diphosphate-dependent enzyme related to transketolase and the pyruvate dehydrogenase E1-beta subunit. DXS is found in bacteria (gene:dxs) and plants (gene:CLA1) which catalyses the thiamine pyrophosphoate-dependent acyloin condensation reaction between carbon atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate (DXP), a precursor in the biosynthetic pathway to isoprenoids, thiamine (vitamin B1), and pyridoxol (vitamin B6). DXS is evolutionary related to TK. This reaction is the first and rate limiting step of the mevalonate-independent pathway for the biosynthesis of isopentyl pyrophosphate, a process undertaken only in fungi, algae and bacteria. DXS differs from related thiamine diphosphate dependent enzymes by virtue of its domain arrangement: the active site is not situated on a chain interface, as seen in related enzymes, but instead resides between two domain faces within the same monomer of a homodimeric complex [PMID:17135236].

 

Reference Protein and Structure

Sequence
Q9RUB5 UniProt (2.2.1.7) IPR005477 (Sequence Homologues) (PDB Homologues)
Biological species
Deinococcus radiodurans R1 (Bacteria) Uniprot
PDB
2o1x - 1-deoxy-D-xylulose 5-phosphate synthase (DXS) from Deinococcus radiodurans (2.9 Å) PDBe PDBsum 2o1x
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 2o1x)
Cofactors
Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.2.1.7)

pyruvate
CHEBI:15361ChEBI
+
D-glyceraldehyde 3-phosphate(2-)
CHEBI:59776ChEBI
+
hydron
CHEBI:15378ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
1-deoxy-D-xylulose 5-phosphate(2-)
CHEBI:57792ChEBI
Alternative enzyme names: 1-deoxy-D-xylulose-5-phosphate pyruvate-lyase (carboxylating), DXP-synthase, 1-deoxyxylulose-5-phosphate synthase,

Enzyme Mechanism

Introduction

Glu373 deprotonates the N1 position of TDP. Charge delocalisation across the conjugated molecule results in concomitant deprotonation of the thiazole ring, activating the cofactor for the next steps in catalysis. The TDP carbanion, stabilised as an ylid, attacks the carbonyl of pyruvate forming a covalently bound intermediate. The oxyanion intermediate collapses to generate carbon dioxide and a TDP-enol intermediate. The carbanion attacks at the glyceraldehyde group to form a tetrahedral oxyanion intermediate. The tetrahedral intermediate collapses releasing the TDP ylid and 1-deoxy-D-xylulose 5-phosphate, the product which enters the isoprenoid biosynthesis pathway [PMID:19778006]. The cofactor is reprotonated to regenerate the active site.

Catalytic Residues Roles

UniProt PDB* (2o1x)
Glu373 Glu373A Activates the thiamine diphosphate cofactor by abstracting a proton, resulting in the production of the active ylid form. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator, electrostatic stabiliser
Lys289, Arg401 Lys289A, Arg401A Act to stabilise the thiamine diphosphate cofactor in its active form. 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, cofactor used, bimolecular nucleophilic addition, overall reactant used, intermediate formation, inferred reaction step, intramolecular elimination, overall product formed, intermediate collapse, charge delocalisation, elimination (not covered by the Ingold mechanisms), unimolecular elimination by the conjugate base, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Xiang S et al. (2007), J Biol Chem, 282, 2676-2682. Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, a Crucial Enzyme for Isoprenoids Biosynthesis. DOI:10.1074/jbc.m610235200. PMID:17135236.
  2. Frank A et al. (2017), Chem Rev, 117, 5675-5703. The Methylerythritol Phosphate Pathway to Isoprenoids. DOI:10.1021/acs.chemrev.6b00537. PMID:27995802.
  3. Brammer LA et al. (2009), Org Lett, 11, 4748-4751. Revealing Substrate Promiscuity of 1-Deoxy-d-xylulose 5-Phosphate Synthase. DOI:10.1021/ol901961q. PMID:19778006.
  4. Shaanan B et al. (2009), FEBS J, 276, 2447-2453. Reaction mechanisms of thiamin diphosphate enzymes: new insights into the role of a conserved glutamate residue. DOI:10.1111/j.1742-4658.2009.06965.x. PMID:19476486.
  5. Jurgenson CT et al. (2009), Annu Rev Biochem, 78, 569-603. The Structural and Biochemical Foundations of Thiamin Biosynthesis. DOI:10.1146/annurev.biochem.78.072407.102340. PMID:19348578.

Step 1. Glu373 deprotonates the N1 position of TDP. Charge delocalisation across the conjugated molecule results in concomitant deprotonation of the thiazole ring, activating the cofactor for the next steps in catalysis. The ionisation of TDP is fast compared to enzymatic turnover, and is therefore not rate determining [PMID:19476486].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu373A activator, hydrogen bond acceptor
Arg401A hydrogen bond donor, electrostatic stabiliser
Lys289A electrostatic stabiliser
Glu373A proton acceptor

Chemical Components

proton transfer, cofactor used

Step 2. The TDP carbanion, stabilised as an ylid, attacks the carbonyl of pyruvate forming a covalently bound intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu373A hydrogen bond donor
Arg401A hydrogen bond donor
Lys289A electrostatic stabiliser
Glu373A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, cofactor used, overall reactant used, intermediate formation, inferred reaction step

Step 3. The oxyanion intermediate collapses to generate carbon dioxide and a TDP-enol intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu373A hydrogen bond donor
Arg401A hydrogen bond donor
Lys289A electrostatic stabiliser

Chemical Components

ingold: intramolecular elimination, cofactor used, intermediate formation, overall product formed, intermediate collapse, charge delocalisation

Step 4. The carbanion attacks at the glyceraldehyde group to form a tetrahedral oxyanion intermediate. Electron delocalisation across the thiazole ring allows for several resonance structures.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu373A hydrogen bond donor
Arg401A hydrogen bond donor
Lys289A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used, intermediate formation, cofactor used

Step 5. The tetrahedral intermediate collapses releasing the TDP ylid and 1-deoxy-D-xylulose 5-phosphate, the product which enters the isoprenoid biosynthesis pathway [PMID:19778006].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg401A hydrogen bond donor
Lys289A electrostatic stabiliser

Chemical Components

elimination (not covered by the Ingold mechanisms), intermediate collapse, cofactor used, overall product formed

Step 6. The cofactor is reprotonated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu373A hydrogen bond donor
Arg401A hydrogen bond donor
Lys289A electrostatic stabiliser
Arg401A electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed

Step 7. The cofactor is reprotonated in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys289A electrostatic stabiliser
Arg401A electrostatic stabiliser
Glu373A electrostatic stabiliser

Chemical Components

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

Step 1. Glu373 deprotonates the N1 position of TDP. Charge delocalisation across the conjugated molecule results in concomitant deprotonation of the thiazole ring, activating the cofactor for the next steps in catalysis. The ionisation of TDP is fast compared to enzymatic turnover, and is therefore not rate determining [PMID:19476486].

Step 2. The TDP carbanion, stabilised as an ylid, attacks the carbonyl of pyruvate forming a covalently bound intermediate.

Step 3. The oxyanion intermediate collapses to generate carbon dioxide and a TDP-enol intermediate.

Step 4. The carbanion attacks at the glyceraldehyde group to form a tetrahedral oxyanion intermediate. Electron delocalisation across the thiazole ring allows for several resonance structures.

Step 5. The tetrahedral intermediate collapses releasing the TDP ylid and 1-deoxy-D-xylulose 5-phosphate, the product which enters the isoprenoid biosynthesis pathway [PMID:19778006].

Step 6. The cofactor is reprotonated.

Step 7. The cofactor is reprotonated in an inferred return step.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Charity Hornby