Squalene synthase

 

Squalene synthase is a member of the Isoprenoid Synthase Type I superfamily. These enzymes catalyse the formation of squalene by the reductive dimerisation of two farnesyl diphosphate molecules in a two-step reaction (2 farnesyl diphosphate --> presqualene diphosphate; presqualene diphosphate + NAD(P)H --> squalene). In the human enzyme, the first half reaction occurs at one end of a large channel (surrounded by five alpha helices runs through the centre of the protein). Then the intermediate is thought to move into an enclosed pocket where the second half reaction occurs. This reaction occurs at the final branch point of the isoprenoid biosynthesis pathway, and is the first committed step in cholesterol biosynthesis.

 

Reference Protein and Structure

Sequence
P37268 UniProt (2.5.1.21) IPR006449 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1ezf - CRYSTAL STRUCTURE OF HUMAN SQUALENE SYNTHASE (2.15 Å) PDBe PDBsum 1ezf
Catalytic CATH Domains
1.10.600.10 CATHdb (see all for 1ezf)
Cofactors
Magnesium(2+) (3) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.5.1.21)

2-trans,6-trans-farnesyl diphosphate(3-)
CHEBI:175763ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
→
squalene
CHEBI:15440ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
Alternative enzyme names: Farnesyltransferase, Presqualene synthase, Presqualene-diphosphate synthase, Farnesyl-diphosphate farnesyltransferase, Squalene synthetase, SQS,

Enzyme Mechanism

Introduction

There is still much debate as to the exact mechanism of this enzyme. It is known that the reaction proceeds via two half reactions, the first of which produces the stable cyclopropylcarbinyl diphosphate intermediate (presqualene diphosphate) from two molecules of 2-trans,6-trans-farnesyl diphosphate. In the second half reaction, the presqualene diphosphate undergoes heterolysis, isomerisation and reduction with NAD(P)H to from squalene. In the absence of NAD(P)H, presqualene diphosphate is accumulated. When NAD(P)H is present, presqualene diphosphate does not dissociate from the enzyme during the synthesis of squalene from farnesyl diphosphate (FPP) [PMID:10677224]. High concentrations of FPP inhibit the production of squalene but not of PSPP [PMID:10677224]. Further discussion revolves around the presence of a single [PMID:10677224], or multiple [PMID:10896663] sites at which the reaction occurs. It is also currently unclear exactly how many magnesium ions are required and bound to the enzyme.

Catalytic Residues Roles

UniProt PDB* (1ezf)
Tyr171 Tyr171(141)A Acts as a general acid/base in the first half reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Arg218, Arg228 Arg218(188)A, Arg228(198)A Acts to stabilise the diphosphate leaving group. promote heterolysis, hydrogen bond donor, electrostatic stabiliser
Phe288 Phe288(258)A Acts to stabilise the reactive carbocation in the second half reaction. Also ensures the correct stereochemical outcome of the reaction. van der waals interaction, steric role, polar/non-polar interaction
*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

intramolecular elimination, proton transfer, charge delocalisation, dephosphorylation, intermediate formation, overall reactant used, overall product formed, bimolecular electrophilic addition, intramolecular nucleophilic addition, cyclisation, heterolysis, intramolecular rearrangement, hydride transfer, bimolecular nucleophilic addition, intermediate terminated, decyclisation, native state of enzyme regenerated

References

  1. Radisky ES et al. (2000), Biochemistry, 39, 1748-1760. Squalene Synthase:  Steady-State, Pre-Steady-State, and Isotope-Trapping Studies†. DOI:10.1021/bi9915014. PMID:10677224.
  2. Liu CI et al. (2014), Acta Crystallogr D Biol Crystallogr, 70, 231-241. Structural insights into the catalytic mechanism of human squalene synthase. DOI:10.1107/S1399004713026230. PMID:24531458.
  3. Blagg BS et al. (2002), J Am Chem Soc, 124, 8846-8853. Recombinant Squalene Synthase. A Mechanism for the Rearrangement of Presqualene Diphosphate to Squalene. DOI:10.1021/ja020411a. PMID:12137537.
  4. Pandit J et al. (2000), J Biol Chem, 275, 30610-30617. Crystal Structure of Human Squalene Synthase: A KEY ENZYME IN CHOLESTEROL BIOSYNTHESIS. DOI:10.1074/jbc.m004132200. PMID:10896663.
  5. Gu P et al. (1998), J Biol Chem, 273, 12515-12525. Function-Structure Studies and Identification of Three Enzyme Domains Involved in the Catalytic Activity in Rat Hepatic Squalene Synthase. DOI:10.1074/jbc.273.20.12515. PMID:9575210.
  6. Poulter CD (1990), Acc Chem Res, 23, 70-77. Biosynthesis of non-head-to-tail terpenes. Formation of 1'-1 and 1'-3 linkages. DOI:10.1021/ar00171a003.

Step 1. Pyrophosphate is eliminated with concomitant deprotonation of Tyr171.

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Catalytic Residues Roles

Residue Roles
Arg228(198)A electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Arg218(188)A electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe288(258)A van der waals interaction
Tyr171(141)A hydrogen bond donor
Tyr171(141)A proton donor

Chemical Components

ingold: intramolecular elimination, proton transfer, charge delocalisation, dephosphorylation, intermediate formation, overall reactant used, overall product formed

Step 2. The second molecule of farnesyl diphosphate initiates an electrophilic attack on the intermediate formed.

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Catalytic Residues Roles

Residue Roles
Arg228(198)A electrostatic stabiliser, hydrogen bond donor
Arg218(188)A electrostatic stabiliser, hydrogen bond donor
Phe288(258)A van der waals interaction
Tyr171(141)A hydrogen bond acceptor

Chemical Components

ingold: bimolecular electrophilic addition, intermediate formation, overall reactant used

Step 3. Tyr171 acts as a base to deprotonate the intermediate, forming the stable intermediate presqualene diphosphate, the product of the first half reaction.

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Catalytic Residues Roles

Residue Roles
Arg228(198)A electrostatic stabiliser, hydrogen bond donor
Arg218(188)A electrostatic stabiliser, hydrogen bond donor
Phe288(258)A van der waals interaction
Tyr171(141)A hydrogen bond acceptor
Tyr171(141)A proton acceptor

Chemical Components

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

Step 4. The substrate undergoes heterolysis. Phe288 essential for the second half reaction

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Catalytic Residues Roles

Residue Roles
Phe288(258)A polar/non-polar interaction, steric role

Chemical Components

heterolysis, intermediate formation, overall product formed

Step 5. A 1,2 sigmatropic rearrangement to produce the cyclobutyl intermediate.

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Catalytic Residues Roles

Residue Roles
Phe288(258)A polar/non-polar interaction, steric role

Chemical Components

intramolecular rearrangement, intermediate formation, cyclisation

Step 6. A 1,2 sigmatropic rearrangement to produce the second cyclopropyl intermediate.

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Catalytic Residues Roles

Residue Roles
Phe288(258)A polar/non-polar interaction, steric role

Chemical Components

intramolecular rearrangement, intermediate formation, cyclisation

Step 7. A hydride transfer from NADP caused the cleavage of the cyclopropyl group to produce squalene.

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Catalytic Residues Roles

Residue Roles
Phe288(258)A polar/non-polar interaction, steric role

Chemical Components

hydride transfer, ingold: intramolecular elimination, ingold: bimolecular nucleophilic addition, intermediate terminated, decyclisation, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Pyrophosphate is eliminated with concomitant deprotonation of Tyr171.

Step 2. The second molecule of farnesyl diphosphate initiates an electrophilic attack on the intermediate formed.

Step 3. Tyr171 acts as a base to deprotonate the intermediate, forming the stable intermediate presqualene diphosphate, the product of the first half reaction.

Step 4. The substrate undergoes heterolysis. Phe288 essential for the second half reaction

Step 5. A 1,2 sigmatropic rearrangement to produce the cyclobutyl intermediate.

Step 6. A 1,2 sigmatropic rearrangement to produce the second cyclopropyl intermediate.

Step 7. A hydride transfer from NADP caused the cleavage of the cyclopropyl group to produce squalene.

Products.

Contributors

Gemma L. Holliday, Charity Hornby