3-deoxy-8-phosphooctulonate synthase

 

3-deoxy-d-manno-s-octulosonate-8-phosphate synthase from Escherichia coli (KDO8P) is responsible for the synthesis of the named product from Phosphenol Pyruvate (PEP) and arabinose-5-phosphate (A5P). It thus plays a role in the synthesis of complex lipids for the gram negative bacterial cell wall, so is a possible target for antibiotics. It displays homology with many PEP utilising enzymes, particularly the metal dependent enzyme DAHP synthase which works by a similar mechanism where a single amino acid change of non-metallo KDO8P changes it to metallo-KDO8P.

 

Reference Protein and Structure

Sequence
P0A715 UniProt (2.5.1.55) IPR006269 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1q3n - Crystal structure of KDO8P synthase in its binary complex with substrate PEP (2.7 Å) PDBe PDBsum 1q3n
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1q3n)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.55)

water
CHEBI:15377ChEBI
+
aldehydo-D-arabinose 5-phosphate(2-)
CHEBI:57693ChEBI
+
phosphonatoenolpyruvate
CHEBI:58702ChEBI
→
3-deoxy-alpha-D-manno-2-octulosonate-8-phosphate(3-)
CHEBI:85985ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: 2-dehydro-3-deoxy-D-octonate-8-phosphate D-arabinose-5-phosphate-lyase (pyruvate-phosphorylating), 2-dehydro-3-deoxy-phosphooctonate aldolase, 2-keto-3-deoxy-8-phosphooctonic synthetase, 3-deoxy-D-manno-octulosonate-8-phosphate synthase, 3-deoxy-D-mannooctulosonate-8-phosphate synthetase, 3-deoxyoctulosonic 8-phosphate synthetase, KDOP synthase, Phospho-2-keto-3-deoxyoctonate aldolase, 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase, KDO-8-phosphate synthetase, KDO-8-P synthase,

Enzyme Mechanism

Introduction

The reaction proceeds through an aldol-like condensation where water activated phosphoenolpyruvate attacks A5P. This forms a bis-phosphorylated tetrahedral intermediate recorded in mass spectrometry experiments. The resulting intermediate then collapses, eliminating a phosphate group. Subsequent sugar cyclisation occurs to form KDO8P. By analogy with the Aquifex aeolicus metallo-KDO8P synthase, water by having hydrogen bonds with the phosphate on PEP increases it's nucleophilicity to attack PEP which in turn is positioned for nucleophilic attack by Lys60. Asp250 and Asn26 hydrogen bond and position A5P for the reaction. Asn26 is thought to mimic the metal ion in metallo-KDO8P enzymes.

Catalytic Residues Roles

UniProt PDB* (1q3n)
Asn26, Lys60, Asp250 Asn26A, Lys60A, Asp250A Position and stabilise the substrates PEP and R5P for the duration of the reaction via hydrogen bonding. 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 nucleophilic substitution, intermediate formation, proton transfer, overall reactant used, intermediate collapse, overall product formed, unimolecular elimination by the conjugate base, intramolecular nucleophilic addition, intermediate terminated

References

  1. Radaev S et al. (2000), J Biol Chem, 275, 9476-9484. Structure and Mechanism of 3-Deoxy-D-manno-octulosonate 8-Phosphate Synthase. DOI:10.1074/jbc.275.13.9476. PMID:10734095.
  2. Harrison AN et al. (2012), Bioorg Med Chem Lett, 22, 907-911. Synthesis and evaluation of tetrahedral intermediate mimic inhibitors of 3-deoxy-d-manno-octulosonate 8-phosphate synthase. DOI:10.1016/j.bmcl.2011.12.025. PMID:22204912.
  3. Tao P et al. (2010), J Inorg Biochem, 104, 1267-1275. Common basis for the mechanism of metallo and non-metallo KDO8P synthases. DOI:10.1016/j.jinorgbio.2010.08.008. PMID:20825995.
  4. Kaustov L et al. (2000), Biochemistry, 39, 14865-14876. Structural and Mechanistic Investigation of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase by Solid-State REDOR NMR†. DOI:10.1021/bi0017172.

Step 1. Water acts as a nucleophile and attacks C2 on PEP, activating C3 PEP to attack the reducing end of A5P. The step passes through an oxocarbenium ion-like transition state.

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Catalytic Residues Roles

Residue Roles
Lys60A electrostatic stabiliser
Asp250A electrostatic stabiliser
Asn26A electrostatic stabiliser, hydrogen bond donor
Asp250A hydrogen bond acceptor
Lys60A hydrogen bond donor

Chemical Components

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

Step 2. Phosphohemiketal intermediate forms. The tetrahedral intermediate collapses to open chain KDO8P.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys60A electrostatic stabiliser
Asp250A electrostatic stabiliser
Asn26A electrostatic stabiliser, hydrogen bond donor
Lys60A hydrogen bond donor
Asp250A hydrogen bond acceptor

Chemical Components

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

Step 3. Sugar condensation to form the ring structure, 3-Deoxy-D-manno-octulosonate phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys60A electrostatic stabiliser
Asp250A electrostatic stabiliser
Asn26A electrostatic stabiliser, hydrogen bond donor
Lys60A hydrogen bond donor
Asp250A hydrogen bond acceptor

Chemical Components

ingold: intramolecular nucleophilic addition, overall product formed, intermediate terminated
Download: Image, Marvin File

Step 1. Water acts as a nucleophile and attacks C2 on PEP, activating C3 PEP to attack the reducing end of A5P. The step passes through an oxocarbenium ion-like transition state.

Step 2. Phosphohemiketal intermediate forms. The tetrahedral intermediate collapses to open chain KDO8P.

Step 3. Sugar condensation to form the ring structure, 3-Deoxy-D-manno-octulosonate phosphate.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Morwenna Hall