5-epi-aristolochene synthase

 

5-epi-aristolochene synthase from tobacco belongs to the family of terpene cyclases and is a member of the I Isoprenoid Synthase Type I superfamily. 5-epi-aristolochene synthase from tobacco (Nicotiana tabacum) converts farnesyl diphosphate into 5-epi-Aristolochene and diphosphate in a magnesium dependent manner. 5-epi-aristolochene is a precursor of the antifungal phytoalexin capsidiol. Based on structural studies of the tobacco enzyme it is believed the terpene is formed via two intermediates, germacrene and a eudesmane carbocation.

 

Reference Protein and Structure

Sequence
Q40577 UniProt (4.2.3.61) IPR008949 (Sequence Homologues) (PDB Homologues)
Biological species
Nicotiana tabacum (common tobacco) Uniprot
PDB
5eat - 5-EPI-ARISTOLOCHENE SYNTHASE FROM NICOTIANA TABACUM WITH SUBSTRATE ANALOG FARNESYL HYDROXYPHOSPHONATE (2.8 Å) PDBe PDBsum 5eat
Catalytic CATH Domains
1.10.600.10 CATHdb (see all for 5eat)
Cofactors
Magnesium(2+) (3) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.2.3.61)

2-trans,6-trans-farnesyl diphosphate(3-)
CHEBI:175763ChEBI
(+)-5-epi-aristolochene
CHEBI:23925ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: 5-epi-aristolochene synthase, Tobacco epiaristolochene synthase, Farnesyl pyrophosphate cyclase, EAS, TEAS,

Enzyme Mechanism

Introduction

A mechanism is proposed based on crystal structures of the enzyme. The first step of the mechanism involves the departure of diphosphate, generating a carbocation intermediate with the positive charge delocalised over C1, C2 and C3. The additional negative charge of the diphosphate is offset by interactions with the three Mg2+ ions, Arg 264 and Arg 441 while the positive charge of the carbocation is stabilised by Thr401 and Thr402 backbone carbonyls and hydroxyl group of Thr403. The carbocation is then poised for attack on C10. Once C1 has been positioned near the p-orbitals of the C10-C11 bond, electrophilic attack at C10 would create a C1-C10 bond and a tertiary carbocation on C11. The quadrupole of Tyr 527 is nicely positioned to stabilise the positive charge on C11. In turn, the newly formed carbocation at C11 substantially increases the acidity of the C12 and C13 methyl protons. Asp 525 then abstracts a proton from C13 (cis methyl group), leading to the formation of a neutral germacrene intermediate. The next step involves proton addition at C6 by the Asp444-Tyr520-Asp525 triad. The carboxyl group of Asp 444 removes the hydroxyl proton from Tyr520. At the same time, the phenolic oxygen at Tyr520 accepts a proton from Asp525. Tyr520 then donates this newly positioned proton to the double bond of germacrene at C6. In a concerted fashion, Tyr520 reaccepts the proton currently on the Asp444 carboxyl group. Concomitant with the C6 protonation by Tyr 520, germacrene undergoes a second ring closure as the p-orbitals on C2 and C7 line up for the formation of the C2-C7 sigma bond. The resulting bicyclic eudesmane carbocation intermediate with the positive charge on C3 is stabilised by the summed dipoles of the Thr401 and Thr402 main chain carbonyls and Thr403 hydroxyl group.
A carbocation centred on C7 must be formed for subsequent reactions. The C2 hydride first migrates to the planar C3 carbocation along the top of the bicyclic ring system leaving a tertiary carbocation with planar geometry at C2. C7 abuts the aromatic face of Trp 273 and stabilisation by this aromatic quadrupole selectively positions the electrophilic centre at C7. The eudesmane carbocation intermediate with the positive charge centred at C2 exists in a conformation that orients the C7-C14 sigma bond parallel to the empty p-orbital on C2. This conformer facilitates the migration of the C14 methyl group from C7 to C2. The conformation of the resulting carbocation with a positive charge positioned on C7 directs a proton on C8 toward the indole ring of Trp 273. The presence of the carbocation centre on C7 greatly increases the acidity of the proton at C8 which may now be removed by Trp 273, giving rise to a positive arenium ion (Trp H1 at residue 273) and the final product, 5-epi-aristolochene.

Catalytic Residues Roles

UniProt PDB* (5eat)
Thr401 (main-C), Thr402 (main-C) Thr401A (main-C), Thr402A (main-C) The carbonyl group stabilises the carbocation intermediates. electrostatic stabiliser, polar interaction
Thr403 Thr403A The hydroxyl group stabilises the carbocation intermediates. electrostatic stabiliser, polar interaction
Asp444 Asp444A Acts as a general acid and base, removing the hydroxyl proton from Tyr 520 and after the protonation of germacrede C6, redonating the proton back to Tyr 520. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Tyr520 Tyr520A Donates a proton, accepted from Asp 525, to germacrene C6 in the formation of the eudesmane carbocation intermediate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Asp525 Asp525A Abstracts a proton from C13 to promote the formation of the germacrene intermediate. It then donates the proton to Tyr 520 to allow it to act as an acid. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Tyr527 Tyr527A The quadrupole of Tyr 527 stabilises the positive charge on C11, created after the formation of the C1-C10 bond. polar interaction, electrostatic stabiliser
Arg441, Arg264 Arg441A, Arg264A Stabilises the additional negative charge on the departing diphosphate in the first step of the reaction. promote heterolysis, hydrogen bond donor, electrostatic stabiliser
Trp273 Trp273A The aromatic quadruple of Trp273 stabilises the electrostatic centre of eudesmane carbocation at C7. It abstracts a proton from C8, forming the final product, 5-epi-aristolochene. hydrogen bond donor, proton acceptor, proton donor, polar interaction, 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

heterolysis, charge delocalisation, dephosphorylation, intermediate formation, overall reactant used, overall product formed, intramolecular electrophilic addition, cyclisation, proton transfer, proton relay, hydride transfer, intramolecular rearrangement, intermediate terminated, inferred reaction step, native state of enzyme regenerated

References

  1. Starks CM et al. (1997), Science, 277, 1815-1820. Structural Basis for Cyclic Terpene Biosynthesis by Tobacco 5-Epi-Aristolochene Synthase. DOI:10.1126/science.277.5333.1815. PMID:9295271.
  2. Faraldos JA et al. (2007), Chembiochem, 8, 1826-1833. Interception of the enzymatic conversion of farnesyl diphosphate to 5-epi-aristolochene by using a fluoro substrate analogue: 1-fluorogermacrene A from (2E,6Z)-6-fluorofarnesyl diphosphate. DOI:10.1002/cbic.200700398. PMID:17886322.

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Arg264A electrostatic stabiliser, promote heterolysis, hydrogen bond donor
Tyr520A hydrogen bond donor
Asp444A hydrogen bond acceptor
Thr401A (main-C) electrostatic stabiliser, polar interaction
Thr402A (main-C) electrostatic stabiliser, polar interaction
Thr403A electrostatic stabiliser, polar interaction

Chemical Components

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

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, hydrogen bond donor
Arg264A electrostatic stabiliser, hydrogen bond donor
Tyr520A hydrogen bond donor
Asp444A hydrogen bond acceptor
Tyr527A polar interaction, electrostatic stabiliser
Thr401A (main-C) electrostatic stabiliser, polar interaction
Thr402A (main-C) electrostatic stabiliser, polar interaction
Thr403A electrostatic stabiliser, polar interaction

Chemical Components

ingold: intramolecular electrophilic addition, charge delocalisation, cyclisation, intermediate formation

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, hydrogen bond donor
Arg264A electrostatic stabiliser, hydrogen bond donor
Tyr520A hydrogen bond donor
Asp444A hydrogen bond acceptor
Asp525A hydrogen bond acceptor
Tyr527A polar interaction, electrostatic stabiliser
Thr401A (main-C) electrostatic stabiliser, polar interaction
Thr402A (main-C) electrostatic stabiliser, polar interaction
Thr403A electrostatic stabiliser, polar interaction
Asp525A proton acceptor

Chemical Components

proton transfer, intermediate formation

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, hydrogen bond donor
Arg264A electrostatic stabiliser, hydrogen bond donor
Tyr520A hydrogen bond donor, hydrogen bond acceptor, proton relay
Asp444A hydrogen bond acceptor
Asp525A hydrogen bond donor
Asp444A proton acceptor
Tyr520A proton acceptor
Asp525A proton donor
Tyr520A proton donor

Chemical Components

proton transfer, proton relay

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, hydrogen bond donor
Arg264A electrostatic stabiliser, hydrogen bond donor
Tyr520A hydrogen bond donor, hydrogen bond acceptor, proton relay
Asp444A hydrogen bond donor
Thr401A (main-C) polar interaction, electrostatic stabiliser
Thr402A (main-C) polar interaction, electrostatic stabiliser
Thr403A polar interaction, electrostatic stabiliser
Tyr520A proton acceptor
Asp444A proton donor
Tyr520A proton donor

Chemical Components

ingold: intramolecular electrophilic addition, proton transfer, cyclisation, intermediate formation, proton relay

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, hydrogen bond donor
Arg264A electrostatic stabiliser, hydrogen bond donor
Tyr520A hydrogen bond donor
Asp444A hydrogen bond acceptor
Thr401A (main-C) polar interaction, electrostatic stabiliser
Thr402A (main-C) polar interaction, electrostatic stabiliser
Thr403A polar interaction, electrostatic stabiliser

Chemical Components

hydride transfer, intermediate formation

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, hydrogen bond donor
Arg264A electrostatic stabiliser, hydrogen bond donor
Tyr520A hydrogen bond donor
Asp444A hydrogen bond acceptor
Trp273A polar interaction, electrostatic stabiliser
Thr401A (main-C) polar interaction, electrostatic stabiliser
Thr402A (main-C) polar interaction, electrostatic stabiliser
Thr403A polar interaction, electrostatic stabiliser

Chemical Components

intramolecular rearrangement, intermediate formation

Catalytic Residues Roles

Residue Roles
Arg441A electrostatic stabiliser, hydrogen bond donor
Arg264A electrostatic stabiliser, hydrogen bond donor
Tyr520A hydrogen bond donor
Asp444A hydrogen bond acceptor
Trp273A hydrogen bond donor
Trp273A proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated

Contributors

Gemma L. Holliday, Mei Leung, James Willey