Pyridoxine 5'-phosphate synthase

 

Pyridoxine 5'-phosphate synthase (PdxJ) catalyses the complicated ring closure reaction between the two acyclic compounds 1-deoxy-D-xylulose-5-phosphate (DXP) and 3-amino-2-oxopropyl phosphate (AAP) to form pyridoxine 5'-phosphate (PNP) and inorganic phosphate. The product of the PdxJ reaction is then oxidised by PdxH to form pyridoxal 5'-phosphate (PLP). PLP is the active form of vitamin B6 (pyridoxine or pyridoxal), a versatile catalyst, acting as a coenzyme in a multitude of reactions, including decarboxylations, deaminations and transaminations.

 

Reference Protein and Structure

Sequence
P0A794 UniProt (2.6.99.2) IPR004569 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1ho1 - CRYSTAL STRUCTURE OF PYRIDOXINE 5'-PHOSPHATE SYNTHASE (2.0 Å) PDBe PDBsum 1ho1
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1ho1)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:2.6.99.2)

1-deoxy-D-xylulose 5-phosphate(2-)
CHEBI:57792ChEBI
+
3-ammonio-2-oxopropyl phosphate(1-)
CHEBI:57279ChEBI
→
water
CHEBI:15377ChEBI
+
hydron
CHEBI:15378ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
pyridoxine 5'-phosphate(2-)
CHEBI:58589ChEBI
Alternative enzyme names: Pyridoxine 5-phosphate phospho lyase, PNP synthase, PdxJ,

Enzyme Mechanism

Introduction

The two substrates react to form a Schiff base. Then His45 deprotonates the C3 of the intermediate, which initiates double bond rearrangement, with the Schiff base acting as an electron sink. The Schiff base reforms, initiating a double bond rearrangement and resulting in the elimination of water, which obtains it's proton from Glu72. Glu72 then deprotonates the remaining hydroxide, which initiates a double bond rearrangement and eliminates phosphate. His12 activates water, which initiates a nucleophilic addition to the newly formed C=C. This causes ring closure, and the resulting oxyanion is protonated by His193. Glu72 is activated by a water molecule. Glu72 deprotonates the C4 carbon, initiating the elimination of hydroxide, which obtains a proton from His12. His193 deprotonates C6, initiating a double bond rearrangement that results in the protonation of O3' by His45.

Catalytic Residues Roles

UniProt PDB* (1ho1)
His193, His12, His45, Glu72 His193(192)A, His12(11)A, His45(44)A, Glu72(71)A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Asn9, Glu153 Asn9(8)A, Glu153(152)A Bind the 3-amino-2-oxopropyl phosphate and act to stabilise the reactive intermediates and transition states during the course of the reaction/ hydrogen bond donor, electrostatic stabiliser
Arg51, Arg47, Thr103 Arg51(50)A, Arg47(46)A, Thr103(102)A Binds 1-deoxy-D-xylulose 5-phosphate and helps stabilise the reactive intermediates and transition states. attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser, increase acidity, increase basicity
*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 addition, proton transfer, overall reactant used, inferred reaction step, intermediate formation, intramolecular elimination, overall product formed, proton relay, dehydration, assisted tautomerisation (not keto-enol), unimolecular elimination by the conjugate base, bimolecular elimination, intramolecular nucleophilic addition, cyclisation, intermediate terminated

References

  1. Garrido-Franco M et al. (2002), J Mol Biol, 321, 601-612. Enzyme–ligand Complexes of Pyridoxine 5′-Phosphate Synthase: Implications for Substrate Binding and Catalysis. DOI:10.1016/s0022-2836(02)00695-2. PMID:12206776.
  2. Mukherjee T et al. (2011), Biochim Biophys Acta, 1814, 1585-1596. Pyridoxal phosphate: biosynthesis and catabolism. DOI:10.1016/j.bbapap.2011.06.018. PMID:21767669.
  3. Yeh JI et al. (2002), Biochemistry, 41, 11649-11657. Multistate Binding in Pyridoxine 5‘-Phosphate Synthase:  1.96 Å Crystal Structure in Complex with 1-Deoxy-d-xylulose Phosphate†. DOI:10.1021/bi026292t. PMID:12269807.

Step 1. The 3-amino-2-oxopropyl phosphate substrate initiates a nucleophilic attack upon the 1-deoxy-D-xylulose 5-phosphate in the first step of a Schiff base formation. The carbonyl oxygen is assumed to deprotonate the amine.

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Catalytic Residues Roles

Residue Roles
Glu72(71)A hydrogen bond donor, electrostatic stabiliser
Thr103(102)A electrostatic stabiliser, hydrogen bond acceptor
Arg47(46)A electrostatic stabiliser, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond donor, electrostatic stabiliser
His12(11)A attractive charge-charge interaction
His45(44)A attractive charge-charge interaction
Arg51(50)A electrostatic stabiliser, hydrogen bond donor, attractive charge-charge interaction
Asn9(8)A hydrogen bond donor, electrostatic stabiliser

Chemical Components

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

Step 2. The newly formed secondary amine initiates a intramolecular elimination of water, which obtains its proton first from Glu72, and then from water. The nature of the proton donor in this step is not entirely clear, it has been assumed to be a water molecule, due to the presence of a water channel in the enzyme [PMID:12206776].

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Catalytic Residues Roles

Residue Roles
Glu72(71)A hydrogen bond donor, proton relay, hydrogen bond acceptor
Thr103(102)A electrostatic stabiliser, hydrogen bond acceptor
Arg47(46)A electrostatic stabiliser, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond donor, electrostatic stabiliser
His12(11)A attractive charge-charge interaction
His45(44)A attractive charge-charge interaction
Arg51(50)A electrostatic stabiliser, hydrogen bond donor, attractive charge-charge interaction
Asn9(8)A hydrogen bond donor, electrostatic stabiliser
Glu72(71)A proton donor, proton acceptor

Chemical Components

ingold: intramolecular elimination, proton transfer, overall product formed, inferred reaction step, intermediate formation, proton relay, dehydration

Step 3. His45 deprotonates the C3 of the intermediate, which initiates double bond rearrangement, with the Schiff base acting as an electron sink.

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Catalytic Residues Roles

Residue Roles
Glu72(71)A hydrogen bond donor
Thr103(102)A electrostatic stabiliser, hydrogen bond acceptor
Arg47(46)A increase basicity, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond donor, electrostatic stabiliser
His12(11)A attractive charge-charge interaction
His45(44)A attractive charge-charge interaction, hydrogen bond acceptor
Arg51(50)A electrostatic stabiliser, hydrogen bond donor, attractive charge-charge interaction
Asn9(8)A hydrogen bond donor, electrostatic stabiliser
His45(44)A proton acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), intermediate formation

Step 4. The Schiff base reforms, initiating a double bond rearrangement and resulting in the elimination of water, which obtains it's proton from Glu72.

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Catalytic Residues Roles

Residue Roles
Glu72(71)A hydrogen bond donor
Thr103(102)A electrostatic stabiliser, hydrogen bond acceptor
Arg47(46)A electrostatic stabiliser, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond donor, electrostatic stabiliser
His12(11)A attractive charge-charge interaction
His45(44)A hydrogen bond donor, attractive charge-charge interaction, electrostatic stabiliser
Arg51(50)A electrostatic stabiliser, hydrogen bond donor, attractive charge-charge interaction
Asn9(8)A hydrogen bond donor, electrostatic stabiliser
Glu72(71)A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate formation, dehydration

Step 5. Glu72 deprotonates the remaining hydroxide, which initiates a double bond rearrangement and eliminates phosphate.

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Catalytic Residues Roles

Residue Roles
Glu72(71)A hydrogen bond acceptor
Thr103(102)A electrostatic stabiliser, hydrogen bond acceptor
Arg47(46)A electrostatic stabiliser, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond donor, electrostatic stabiliser
His12(11)A attractive charge-charge interaction
His45(44)A hydrogen bond donor, attractive charge-charge interaction, electrostatic stabiliser
Arg51(50)A electrostatic stabiliser, hydrogen bond donor, attractive charge-charge interaction
Asn9(8)A hydrogen bond donor, electrostatic stabiliser
Glu72(71)A proton acceptor

Chemical Components

ingold: bimolecular elimination, intermediate formation, overall product formed

Step 6. His12 activates water, which initiates a nucleophilic addition to the newly formed C=C. This causes ring closure, and the resulting oxyanion is protonated by His193. Glu72 is activated by a water molecule. It is suggested that one of the histidines at the top of the active site are responsible for the activation of water in this step. His12 and His52 are both candidates [PMID:12206776].

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Catalytic Residues Roles

Residue Roles
Glu72(71)A hydrogen bond donor
Arg47(46)A electrostatic stabiliser, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond donor
His12(11)A hydrogen bond acceptor, attractive charge-charge interaction
His45(44)A hydrogen bond donor, attractive charge-charge interaction, electrostatic stabiliser
Arg51(50)A electrostatic stabiliser, increase basicity, hydrogen bond donor, attractive charge-charge interaction
Asn9(8)A hydrogen bond donor, electrostatic stabiliser
His193(192)A proton donor
Glu72(71)A proton donor
His12(11)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, ingold: intramolecular nucleophilic addition, proton transfer, cyclisation, intermediate formation

Step 7. Glu72 deprotonates the C4 carbon, initiating the elimination of hydroxide, which obtains a proton from His12.

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Catalytic Residues Roles

Residue Roles
Glu72(71)A hydrogen bond acceptor
Arg47(46)A electrostatic stabiliser, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond acceptor
His12(11)A hydrogen bond donor, attractive charge-charge interaction
His45(44)A hydrogen bond donor, attractive charge-charge interaction, electrostatic stabiliser
Arg51(50)A electrostatic stabiliser, increase acidity, hydrogen bond donor, attractive charge-charge interaction
Asn9(8)A hydrogen bond donor, electrostatic stabiliser
Glu72(71)A proton acceptor
His12(11)A proton donor

Chemical Components

ingold: bimolecular elimination, proton transfer, intermediate formation, overall product formed, dehydration

Step 8. His193 deprotonates C6, initiating a double bond rearrangement that results in the protonation of O3' by His45.

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Catalytic Residues Roles

Residue Roles
Arg47(46)A increase acidity, attractive charge-charge interaction
Glu153(152)A hydrogen bond acceptor, electrostatic stabiliser
His193(192)A hydrogen bond acceptor
His12(11)A hydrogen bond donor, attractive charge-charge interaction
His45(44)A hydrogen bond donor, attractive charge-charge interaction
Arg51(50)A electrostatic stabiliser, hydrogen bond donor, attractive charge-charge interaction
His193(192)A proton acceptor
His45(44)A proton donor

Chemical Components

proton transfer, intermediate terminated, overall product formed
Download: Image, Marvin File

Step 1. The 3-amino-2-oxopropyl phosphate substrate initiates a nucleophilic attack upon the 1-deoxy-D-xylulose 5-phosphate in the first step of a Schiff base formation. The carbonyl oxygen is assumed to deprotonate the amine.

Step 2. The newly formed secondary amine initiates a intramolecular elimination of water, which obtains its proton first from Glu72, and then from water. The nature of the proton donor in this step is not entirely clear, it has been assumed to be a water molecule, due to the presence of a water channel in the enzyme [PMID:12206776].

Step 3. His45 deprotonates the C3 of the intermediate, which initiates double bond rearrangement, with the Schiff base acting as an electron sink.

Step 4. The Schiff base reforms, initiating a double bond rearrangement and resulting in the elimination of water, which obtains it's proton from Glu72.

Step 5. Glu72 deprotonates the remaining hydroxide, which initiates a double bond rearrangement and eliminates phosphate.

Step 6. His12 activates water, which initiates a nucleophilic addition to the newly formed C=C. This causes ring closure, and the resulting oxyanion is protonated by His193. Glu72 is activated by a water molecule. It is suggested that one of the histidines at the top of the active site are responsible for the activation of water in this step. His12 and His52 are both candidates [PMID:12206776].

Step 7. Glu72 deprotonates the C4 carbon, initiating the elimination of hydroxide, which obtains a proton from His12.

Step 8. His193 deprotonates C6, initiating a double bond rearrangement that results in the protonation of O3' by His45.

Products.

Contributors

Gemma L. Holliday, Charity Hornby