Pentalenene synthase

 

Pentalenene synthase is structurally related to farnesyl diphosphate synthase, in accordance with the hypothesis that successive enzymes on a biosynthetic pathway are often evolutionarily related. Pentalene is a sesquiterpene that is a precursor for the pentalenolactone family of antibiotics. The sequiterpene family of secondary metabolites is a very large one, being secreted by plants, fungi and some microorganisms.

 

Reference Protein and Structure

Sequence
Q55012 UniProt (4.2.3.7) IPR034686 (Sequence Homologues) (PDB Homologues)
Biological species
Streptomyces exfoliatus (Bacteria) Uniprot
PDB
1ps1 - PENTALENENE SYNTHASE (2.6 Å) PDBe PDBsum 1ps1
Catalytic CATH Domains
1.10.600.10 CATHdb (see all for 1ps1)
Cofactors
Magnesium(2+) (3) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.2.3.7)

2-trans,6-trans-farnesyl diphosphate(3-)
CHEBI:175763ChEBI
→
pentalenene
CHEBI:17251ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: Pentalenene synthetase,

Enzyme Mechanism

Introduction

Originally based on QM/MM calculations, this is now the more generally accepted mechanism. In this mechanism, the initial cyclisation of FPP to the (E,E)-humulyl cation is followed by a 1,2-hydride shift, either directly or via a deprotonation–protonation sequence. The resulting protoilludyl cation then reactions in a dyotropic rearrangement. The product of this penultimate step is then deprotonated to form the final product. The general acid/base is assumed to be the pyrophosphate group.

Catalytic Residues Roles

UniProt PDB* (1ps1)
Ser223, Glu227, Asn219 Ser223A, Glu227A, Asn219A Forms part of the magnesium 3 binding site. metal ligand
Trp308, His309, Phe76, Phe77 Trp308A, His309A, Phe76A, Phe77A F76 and F77 are well placed so as to interact with positive charge at C-1, C-2, and C-3 of the farnesyl residue and derived intermediates and act to stabilise these charges through quadrupole-charge interactions. van der waals interaction, electrostatic stabiliser
Arg230, Arg157, Arg173, Lys226 Arg230A, Arg157A, Arg173A, Lys226A Stabilises the pyrophosphate leaving group. electrostatic stabiliser
Asp84, Asp80 Asp84A, Asp80A Forms part of the magnesium 1 and 2 binding sites. metal ligand
*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

heterolysis, overall reactant used, charge delocalisation, overall product formed, dephosphorylation, intermediate formation, intramolecular electrophilic addition, cyclisation, hydride transfer, hydrogen transfer, intramolecular rearrangement, proton transfer, bimolecular elimination

References

  1. Dickschat JS (2016), Nat Prod Rep, 33, 87-110. Bacterial terpene cyclases. DOI:10.1039/c5np00102a. PMID:26563452.
  2. Pemberton TA et al. (2016), J Antibiot (Tokyo), 69, 486-493. General base-general acid catalysis by terpenoid cyclases. DOI:10.1038/ja.2016.39. PMID:27072285.
  3. Lodewyk MW et al. (2014), Org Biomol Chem, 12, 887-894. Pentalenene formation mechanisms redux. DOI:10.1039/c3ob42005a. PMID:24326700.
  4. Zu L et al. (2012), J Am Chem Soc, 134, 11369-11371. Effect of Isotopically Sensitive Branching on Product Distribution for Pentalenene Synthase: Support for a Mechanism Predicted by Quantum Chemistry. DOI:10.1021/ja3043245. PMID:22738258.
  5. Tantillo DJ (2011), Nat Prod Rep, 28, 1035-1053. Biosynthesis via carbocations: Theoretical studies on terpene formation. DOI:10.1039/c1np00006c. PMID:21541432.
  6. Gutta P et al. (2006), J Am Chem Soc, 128, 6172-6179. Theoretical Studies on Farnesyl Cation Cyclization:  Pathways to Pentalenene. DOI:10.1021/ja058031n. PMID:16669687.
  7. Seemann M et al. (2002), J Am Chem Soc, 124, 7681-7689. Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis. PMID:12083921.
  8. Lesburg CA et al. (1997), Science, 277, 1820-1824. Crystal Structure of Pentalenene Synthase: Mechanistic Insights on Terpenoid Cyclization Reactions in Biology. DOI:10.1126/science.277.5333.1820. PMID:9295272.

Step 1. The substrate undergoes heterolysis. The diphosphate product remains associated with the active site, and the cabocation is delocalised over the three terminal carbon atoms of the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg230A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg173A electrostatic stabiliser
Arg157A electrostatic stabiliser
Ser223A metal ligand
Asn219A metal ligand
Glu227A metal ligand
Asp80A metal ligand
Asp84A metal ligand
Phe76A van der waals interaction
Phe77A steric role, van der waals interaction
Asn219A steric role, polar/non-polar interaction
Trp308A van der waals interaction

Chemical Components

heterolysis, overall reactant used, charge delocalisation, overall product formed, dephosphorylation, intermediate formation

Step 2. The terminal double bond adds to the terminal carbocation in an intramolecular electrophilic addition resulting in a cyclic intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser223A metal ligand
Asn219A metal ligand
Glu227A metal ligand
Asp80A metal ligand
Asp84A metal ligand
Arg230A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg173A electrostatic stabiliser
Arg157A electrostatic stabiliser
Phe76A electrostatic stabiliser, van der waals interaction
Phe77A electrostatic stabiliser, van der waals interaction
Asn219A electrostatic stabiliser, polar/non-polar interaction
Trp308A electrostatic stabiliser, van der waals interaction

Chemical Components

ingold: intramolecular electrophilic addition, cyclisation, intermediate formation

Step 3. The cyclic intermediate undergoes a [1,2]-hydride shift.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe76A electrostatic stabiliser
Phe77A electrostatic stabiliser
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Trp308A electrostatic stabiliser
His309A electrostatic stabiliser
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand

Chemical Components

hydride transfer

Step 4. Double bond rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe76A electrostatic stabiliser
Phe77A electrostatic stabiliser
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Trp308A electrostatic stabiliser
His309A electrostatic stabiliser
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand

Chemical Components

ingold: intramolecular electrophilic addition, cyclisation

Step 5. The substrate undergoes a dyotropic rearrangement. This is an unusual elementary step in this terpene cyclisation, especially because the cationic centre seems to be not involved and rather plays a role as a spectator, but certainly the neighbouring migrating C–C-bond of the cyclobutane is substantially weakened and thus more reactive due to the presence of the cation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand
Phe76A electrostatic stabiliser
Phe77A electrostatic stabiliser
Arg173A electrostatic stabiliser
Asn219A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Trp308A electrostatic stabiliser
His309A electrostatic stabiliser

Chemical Components

hydrogen transfer, intramolecular rearrangement

Step 6. The phosphate acts as a general acid/base, abstracting the proton from the final intermediate and generating the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe76A electrostatic stabiliser
Phe77A electrostatic stabiliser
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Trp308A electrostatic stabiliser
His309A electrostatic stabiliser
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand

Chemical Components

proton transfer, ingold: bimolecular elimination
Download: Image, Marvin File

Step 1. The substrate undergoes heterolysis. The diphosphate product remains associated with the active site, and the cabocation is delocalised over the three terminal carbon atoms of the intermediate.

Step 2. The terminal double bond adds to the terminal carbocation in an intramolecular electrophilic addition resulting in a cyclic intermediate.

Step 3. The cyclic intermediate undergoes a [1,2]-hydride shift.

Step 4. Double bond rearrangement.

Step 5. The substrate undergoes a dyotropic rearrangement. This is an unusual elementary step in this terpene cyclisation, especially because the cationic centre seems to be not involved and rather plays a role as a spectator, but certainly the neighbouring migrating C–C-bond of the cyclobutane is substantially weakened and thus more reactive due to the presence of the cation.

Step 6. The phosphate acts as a general acid/base, abstracting the proton from the final intermediate and generating the final product.

Products.

Introduction

Farnesyl diphosphate is thought to undergo ionisation and electrophilic attack of the incipient allyl cation pyrophosphate pair on the distal pi bond (see PMID:9295272). Metal-triggered substrate ionisation initiates catalysis. The initial step in the reaction is likely to be a cyclisation of farnesyl diphosphate to form humulene. The most likely catalytic base is thought to be the phosphate anion.

Catalytic Residues Roles

UniProt PDB* (1ps1)
Ser223, Glu227, Asn219 Ser223A, Glu227A, Asn219A Forms part of the magnesium 3 binding site. metal ligand
Trp308, His309, Phe76, Phe77 Trp308A, His309A, Phe76A, Phe77A F76 and F77 are well placed so as to interact with positive charge at C-1, C-2, and C-3 of the farnesyl residue and derived intermediates and act to stabilise these charges through quadrupole-charge interactions. van der waals interaction, electrostatic stabiliser
Arg230, Arg157, Arg173, Lys226 Arg230A, Arg157A, Arg173A, Lys226A Stabilises the pyrophosphate leaving group. electrostatic stabiliser
Asp84, Asp80 Asp84A, Asp80A Forms part of the magnesium 1 and 2 binding sites. metal ligand
*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

heterolysis, overall reactant used, charge delocalisation, overall product formed, dephosphorylation, intermediate formation, intramolecular electrophilic addition, cyclisation, hydride transfer, intramolecular nucleophilic substitution, intramolecular nucleophilic addition, proton transfer, intermediate terminated

References

  1. Lesburg CA et al. (1997), Science, 277, 1820-1824. Crystal Structure of Pentalenene Synthase: Mechanistic Insights on Terpenoid Cyclization Reactions in Biology. DOI:10.1126/science.277.5333.1820. PMID:9295272.
  2. Pemberton TA et al. (2016), J Antibiot (Tokyo), 69, 486-493. General base-general acid catalysis by terpenoid cyclases. DOI:10.1038/ja.2016.39. PMID:27072285.
  3. Zu L et al. (2012), J Am Chem Soc, 134, 11369-11371. Effect of Isotopically Sensitive Branching on Product Distribution for Pentalenene Synthase: Support for a Mechanism Predicted by Quantum Chemistry. DOI:10.1021/ja3043245. PMID:22738258.
  4. Seemann M et al. (2002), J Am Chem Soc, 124, 7681-7689. Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis. PMID:12083921.
  5. Seemann M et al. (1999), J Am Chem Soc, 121, 591-592. Pentalenene Synthase. Histidine-309 Is Not Required for Catalytic Activity. DOI:10.1021/ja983657h.
  6. Cane DE et al. (1994), Biochemistry, 33, 5846-5857. Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319. PMID:8180213.

Step 1. The substrate undergoes heterolysis. The diphosphate product remains associated with the active site, and the cabocation is delocalised over the three terminal carbon atoms of the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe76A van der waals interaction
Phe77A steric role, van der waals interaction
Asn219A steric role, polar/non-polar interaction
Trp308A van der waals interaction
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser

Chemical Components

heterolysis, overall reactant used, charge delocalisation, overall product formed, dephosphorylation, intermediate formation

Step 2. The terminal double bond adds to the terminal carbocation in an intramolecular electrophilic addition resulting in a cyclic intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe76A electrostatic stabiliser, van der waals interaction
Phe77A electrostatic stabiliser, van der waals interaction
Asn219A electrostatic stabiliser, polar/non-polar interaction
Trp308A electrostatic stabiliser, van der waals interaction
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand

Chemical Components

ingold: intramolecular electrophilic addition, cyclisation, intermediate formation

Step 3. The intermediate undergoes a [1,2]-hydride shift.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His309A hydrogen bond acceptor
Phe76A electrostatic stabiliser, van der waals interaction
Phe77A electrostatic stabiliser, van der waals interaction
Asn219A electrostatic stabiliser, polar/non-polar interaction
Trp308A electrostatic stabiliser, van der waals interaction
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand

Chemical Components

intermediate formation, hydride transfer

Step 4. In a second intramolecular electrophilic addition, a double bond adds across the cycle forming a five membered ring and a new carbocation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His309A hydrogen bond donor
Phe76A van der waals interaction
Phe77A van der waals interaction
Asn219A polar/non-polar interaction
Trp308A van der waals interaction
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand

Chemical Components

ingold: intramolecular electrophilic addition, cyclisation, intermediate formation

Step 5. In a third intramolecular electrophilic addition, the remaining double bond adds across the cycle to form the three five membered ring motif of pentalenene, this also causes a [1,2]-hydride shift and the carbocation shifting to a different carbon atom

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe76A electrostatic stabiliser, van der waals interaction
Phe77A electrostatic stabiliser, van der waals interaction
Asn219A electrostatic stabiliser, polar/non-polar interaction
Trp308A electrostatic stabiliser, van der waals interaction
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser

Chemical Components

ingold: intramolecular nucleophilic substitution, hydride transfer, ingold: intramolecular nucleophilic addition, cyclisation, intermediate formation

Step 6. The phosphate group deprotonates the carbon adjacent to the new carbocation, forming the final double bond of pentalenene.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His309A hydrogen bond acceptor
Phe76A electrostatic stabiliser, van der waals interaction
Phe77A electrostatic stabiliser, van der waals interaction
Asn219A electrostatic stabiliser, polar/non-polar interaction
Trp308A electrostatic stabiliser, van der waals interaction
Arg157A electrostatic stabiliser
Arg173A electrostatic stabiliser
Lys226A electrostatic stabiliser
Arg230A electrostatic stabiliser
Asp84A metal ligand
Asp80A metal ligand
Glu227A metal ligand
Asn219A metal ligand
Ser223A metal ligand

Chemical Components

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

Step 1. The substrate undergoes heterolysis. The diphosphate product remains associated with the active site, and the cabocation is delocalised over the three terminal carbon atoms of the intermediate.

Step 2. The terminal double bond adds to the terminal carbocation in an intramolecular electrophilic addition resulting in a cyclic intermediate.

Step 3. The intermediate undergoes a [1,2]-hydride shift.

Step 4. In a second intramolecular electrophilic addition, a double bond adds across the cycle forming a five membered ring and a new carbocation.

Step 5. In a third intramolecular electrophilic addition, the remaining double bond adds across the cycle to form the three five membered ring motif of pentalenene, this also causes a [1,2]-hydride shift and the carbocation shifting to a different carbon atom

Step 6. The phosphate group deprotonates the carbon adjacent to the new carbocation, forming the final double bond of pentalenene.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Alex Gutteridge, Craig Porter