M-CSA Mechanism and Catalytic Site Atlas

Epi-isozizaene synthase

epi-isozizaene synthase is a member of the the Isoprenoid Synthase Type I superfamily. It catalyses the cyclisation of farnesyl diphosphate (FPP) to the sesquiterpene epi-isozizaene which is part of the sesquiterpene biosynthesis pathway.

Reference Protein and Structure

Sequence: Q9K499 (4.2.3.37) (Sequence Homologues) (PDB Homologues)
Biological species: Streptomyces coelicolor A3(2) (Bacteria)
PDB: 3kb9 - Epi-isozizaene synthase: Complex with Mg, inorganic pyrophosphate and benzyl triethyl ammonium cation (1.598 Å)
Catalytic CATH Domains: 1.10.600.10 (see all for 3kb9)
Cofactors: Magnesium(2+) (3) Metal MACiE

Click To Show Structure

Enzyme Reaction (EC:4.2.3.37)

2-trans,6-trans-farnesyl diphosphate(3-)

CHEBI:175763

→

diphosphate(3-)

CHEBI:33019

(+)-epi-isozizaene

CHEBI:51458

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: SCO5222 protein,

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

Summary
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Step 8
Products
All Steps

Introduction

The binding of the substrate causes a conformation change in the active site, which promotes the elimination of the pyrophosphate. The pyrophosphate attacks the C3 cationic carbon in a nucleophilic addition reaction. Intramolecular electrophilic addition across the C1-C6 bond to form the six-membered ring with concomitant elimination of the pyrophosphate and initiates a 1,2-Hydride shift. Spirocyclisation occurs to form the five and six membered ring intermediate. Intramolecular electrophilic addition to form the second six-membered ring which then contracts to a five membered ring. The pyrophosphate deprotonates the substrate, which initiates the final methyl migration.

Catalytic Residues Roles

UniProt	PDB* (3kb9)
Phe95, Phe96, Phe198	Phe95(116)A, Phe96(117)A, Phe198(219)A	Act to stabilise the positive carbocations formed during the course of the reaction. Also involved in ensuring the correct stereoisomers are formed.	polar/non-polar interaction, steric role, electrostatic stabiliser
Arg338	Arg338(359)A	Forms hydrogen bond with D219 that facilitates active site closure; Hydrogen bonds to PPi anion	promote heterolysis, 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

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

References

Aaron JA et al. (2010), Biochemistry, 49, 1787-1797. Structure of Epi-Isozizaene Synthase fromStreptomyces coelicolorA3(2), a Platform for New Terpenoid Cyclization Templates,. DOI:10.1021/bi902088z. PMID:20131801.
Rabe P et al. (2016), Chembiochem, 17, 1333-1337. Position-Specific Mass Shift Analysis: A Systematic Method for Investigating the EI-MS Fragmentation Mechanism of epi-Isozizaene. DOI:10.1002/cbic.201600237. PMID:27123899.
Lin X et al. (2006), J Am Chem Soc, 128, 6022-6023. Genome Mining inStreptomycescoelicolor: Molecular Cloning and Characterization of a New Sesquiterpene Synthase. DOI:10.1021/ja061292s. PMID:16669656.

Step 1. The binding of the substrate causes a conformation change in the active site, which promotes the elimination of the pyrophosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

ingold: intramolecular elimination, charge delocalisation, dephosphorylation, intermediate formation, overall reactant used

Step 2. The pyrophosphate attacks the C3 cationic carbon in a nucleophilic addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

ingold: bimolecular nucleophilic addition, charge delocalisation, intermediate formation

Step 3. Rotation about the newly formed C2-C3 bond generates the correpsonding cicoid conformer, which readily undergoes ionisation and cyclisation (across the C1-C6 bond) to form the bisabolyl cation [PMID:16669656] with concomitant dephosphorylation.

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Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

ingold: intramolecular electrophilic substitution, intermediate formation, cyclisation, dephosphorylation

Step 4. A 1,2-Hydride shift.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

hydride transfer, intramolecular rearrangement, intermediate formation

Step 5. Spirocyclisation to form the five and six membered ring acorenyl intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

intramolecular rearrangement, intermediate formation, cyclisation

Step 6. Intramolecular electrophilic addition to form the second six-membered ring.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

ingold: intramolecular electrophilic addition, intermediate formation, cyclisation

Step 7. The six-membered ring contracts to a five membered ring.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

intramolecular rearrangement, intermediate formation

Step 8. The pyrophosphate deprotonates the substrate, which initiates the final methyl migration.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Arg338(359)A	electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Phe95(116)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe96(117)A	steric role, electrostatic stabiliser, polar/non-polar interaction
Phe198(219)A	steric role, electrostatic stabiliser, polar/non-polar interaction

Chemical Components

proton transfer, intramolecular rearrangement, intermediate terminated, overall product formed, native state of enzyme regenerated

Download: Image, Marvin File

Step 1. The binding of the substrate causes a conformation change in the active site, which promotes the elimination of the pyrophosphate.

Step 2. The pyrophosphate attacks the C3 cationic carbon in a nucleophilic addition reaction.

Step 4. A 1,2-Hydride shift.

Step 5. Spirocyclisation to form the five and six membered ring acorenyl intermediate.

Step 6. Intramolecular electrophilic addition to form the second six-membered ring.

Step 7. The six-membered ring contracts to a five membered ring.

Step 8. The pyrophosphate deprotonates the substrate, which initiates the final methyl migration.

Products.

Contributors

Gemma L. Holliday, James Willey