Inositol-3-phosphate synthase

 

Inositol-3-phosphate synthase catalyses the conversion of D-glucose 6-phosphate to 1L-myo-inositol-1-phosphate, the first committed step in the production of all inositol-containing compounds, including phospholipids, either directly or by salvage. It requires NAD+, which dehydrogenates the -CHOH- group to -CO- at C-5 of the glucose 6-phosphate, making C-6 into an active methylene, able to condense with the -CHO at C-1. Finally, the enzyme-bound NADH reconverts C-5 into the -CHOH- form.

 

Reference Protein and Structure

Sequence
P11986 UniProt (5.5.1.4) IPR002587 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1rm0 - Crystal Structure of Myo-Inositol 1-Phosphate Synthase From Saccharomyces cerevisiae In Complex With NAD+ and 2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate (2.05 Å) PDBe PDBsum 1rm0
Catalytic CATH Domains
3.30.360.10 CATHdb 3.40.50.720 CATHdb (see all for 1rm0)
Cofactors
Nadh(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:5.5.1.4)

D-glucopyranose 6-phosphate(2-)
CHEBI:61548ChEBI
→
1D-myo-inositol 3-phosphate(2-)
CHEBI:58401ChEBI
Alternative enzyme names: Myo-inositol-1-phosphate synthase, D-glucose 6-phosphate cycloaldolase, Glucocycloaldolase, Glucose 6-phosphate cyclase, Glucose-6-phosphate inositol monophosphate cycloaldolase, Inositol 1-phosphate synthatase, Inositol 1-phosphate synthetase, 1L-myo-inositol-1-phosphate lyase (isomerizing),

Enzyme Mechanism

Introduction

The substrate is oxidised at the C5' by NAD+, with direct hydride transfer from C5 to C4 of the nicotinamide ring in concert with proton loss from the C5' hydroxyl group of D-glucose-6-phosphate. This proton is transferred to the Lys369 side chain. The pro-R hydrogen of C6 is eliminated. Evidence from crystallographic data suggests the phosphate-mono ester acts as the base in the enolisation step. The developing negative charge on the enolate oxygen is stabilised by two lysine residues, Lys369 and Lys489. In this aldol condensation step either the monophosphate or lys412 acts as a general acid to O1 in the cyclisation step. The last mechanistic step is reduction by NADPH. The hydride that was transferred in the first step is returned to the C5 position of the intermediate myo-2-inose 1-phosphate. The active site is regenerated on protonation.

Catalytic Residues Roles

UniProt PDB* (1rm0)
Lys412 Lys412A Acts as a general acid/base involved in ring opening and closing. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator, electrostatic stabiliser
Lys489, Lys369 Lys489A, Lys369A Acts as a general acid/base. Also helps to stabilise the negative charge on the enolate oxygen. proton acceptor, hydrogen bond donor, electrostatic stabiliser, proton donor
Asp320 Asp320A Important for the stabilisation of the resulting positive charge by acting as a hydrogen bond acceptor to the general acid/base Lys369. hydrogen bond acceptor, 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

hydride transfer, proton transfer, intermediate formation, overall reactant used, cofactor used, assisted keto-enol tautomerisation, intramolecular nucleophilic addition, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated, inferred reaction step

References

  1. Jin X et al. (2004), J Biol Chem, 279, 13889-13895. The Structure of the 1L-myo-inositol-1-phosphate Synthase-NAD+-2-deoxy-D-glucitol 6-(E)-Vinylhomophosphonate Complex Demands a Revision of the Enzyme Mechanism. DOI:10.1074/jbc.m308986200. PMID:14684747.
  2. Seelan RS et al. (2009), J Biol Chem, 284, 9443-9457. Identification of myo-Inositol-3-phosphate Synthase Isoforms: CHARACTERIZATION, EXPRESSION, AND PUTATIVE ROLE OF A 16-kDa  c ISOFORM. DOI:10.1074/jbc.m900206200. PMID:19188364.
  3. Stein AJ et al. (2002), J Biol Chem, 277, 9484-9491. The Crystal Structure and Mechanism of 1-L-myo-Inositol- 1-phosphate Synthase. DOI:10.1074/jbc.m109371200. PMID:11779862.

Step 1. The substrate is oxidised at the C5' by NAD+, with direct hydride transfer from C5 to C4 of the nicotinamide ring in concert with proton loss from the C5' hydroxyl group of D-glucose-6-phosphate. This proton is transferred to the Lys369 side chain.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys412A hydrogen bond donor
Asp320A electrostatic stabiliser, hydrogen bond acceptor
Lys369A activator, hydrogen bond acceptor
Lys489A hydrogen bond donor, electrostatic stabiliser
Lys369A proton acceptor

Chemical Components

hydride transfer, proton transfer, intermediate formation, overall reactant used, cofactor used

Step 2. The pro-R hydrogen of C6 is eliminated. Evidence from crystallographic data suggests the phosphate-mono ester acts as the base in the enolisation step. The developing negative charge on the enolate oxygen is stabilised by two lysine residues, Lys369 and Lys489.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys412A hydrogen bond donor
Asp320A electrostatic stabiliser, hydrogen bond acceptor
Lys369A electrostatic stabiliser, hydrogen bond donor
Lys489A electrostatic stabiliser, hydrogen bond donor

Chemical Components

assisted keto-enol tautomerisation, intermediate formation

Step 3. In this aldol condensation step either the monophosphate or Lys412 acts as a general acid to O1 in the cyclisation step. The side chain of Lys373 is thought to aid in stabilising the O1 negative charge [PMID:14684747].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys412A activator, electrostatic stabiliser, hydrogen bond donor
Asp320A electrostatic stabiliser, hydrogen bond acceptor
Lys369A electrostatic stabiliser, hydrogen bond donor
Lys489A electrostatic stabiliser, hydrogen bond donor
Lys412A proton donor

Chemical Components

assisted keto-enol tautomerisation, ingold: intramolecular nucleophilic addition, proton transfer, intermediate formation

Step 4. The last mechanistic step is reduction by NADPH. The hydride that was transferred in the first step is returned to the C5 position of the intermediate myo-2-inose 1-phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys412A hydrogen bond acceptor
Asp320A electrostatic stabiliser, hydrogen bond acceptor
Lys369A activator, hydrogen bond donor
Lys489A electrostatic stabiliser, hydrogen bond donor
Lys489A proton donor

Chemical Components

hydride transfer, proton transfer, overall product formed, native state of cofactor regenerated, cofactor used

Step 5. The active site is regenerated on protonation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys412A activator, hydrogen bond acceptor
Asp320A hydrogen bond acceptor
Lys369A hydrogen bond donor
Lys489A hydrogen bond donor, proton acceptor
Lys412A proton acceptor
Lys369A proton donor

Chemical Components

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

Step 1. The substrate is oxidised at the C5' by NAD+, with direct hydride transfer from C5 to C4 of the nicotinamide ring in concert with proton loss from the C5' hydroxyl group of D-glucose-6-phosphate. This proton is transferred to the Lys369 side chain.

Step 2. The pro-R hydrogen of C6 is eliminated. Evidence from crystallographic data suggests the phosphate-mono ester acts as the base in the enolisation step. The developing negative charge on the enolate oxygen is stabilised by two lysine residues, Lys369 and Lys489.

Step 3. In this aldol condensation step either the monophosphate or Lys412 acts as a general acid to O1 in the cyclisation step. The side chain of Lys373 is thought to aid in stabilising the O1 negative charge [PMID:14684747].

Step 4. The last mechanistic step is reduction by NADPH. The hydride that was transferred in the first step is returned to the C5 position of the intermediate myo-2-inose 1-phosphate.

Step 5. The active site is regenerated on protonation.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday