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CSA entry for 5eat
Original Entry
Title:
Isoprenoid synthase
Compound:
5-epi-aristolochene synthase
Mutant:
No
UniProt/Swiss-Prot:
Q40577-5EAS_TOBAC
EC Class:
4.2.3.9
Other CSA Entries:
Overview of all sites for 5eat
Homologues of 5eat
Entries for UniProt/Swiss-Prot: Q40577
Entries for EC: 4.2.3.9
Other Databases:
PDB entry: 5eat
PDBsum entry: 5eat
UniProt/Swiss-Prot: Q40577
IntEnz entry: 4.2.3.9
Literature Report:
Introduction:
Tobacco 5-epi-aristolochene synthase(TEAS) converts farnesyl diphosphate to 5-epi-aristolochene, a precursor of the antifungal phytoalexin capsidiol.
Mechanism:
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.
Sites:

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
851 0
ElectrostaticTransition state
It stabilises the additional negative charge on the departing diphosphate in the first step of the reaction.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
852 0
ElectrostaticTransition state
It stabilises the additional negative charge on the departing diphosphate in the first step of the reaction.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
853 0
ElectrostaticTransition state
It stabilises the additional negative charge on the departing diphosphate in the first step of the reaction.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ARGA 264 264Sidechain
ElectrostaticTransition state
It stabilises the additional negative charge on the departing diphosphate in the first step of the reaction.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
TRPA 273 273Sidechain
Acid/baseSubstrate
ElectrostaticTransition state
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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Mutagenesis of residue
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
THRA 401 401Backbone carbonyl
ElectrostaticTransition state
Its carbonyl group stabilises the carbocation intermediates.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
THRA 402 402Backbone carbonyl
ElectrostaticTransition state
Its carbonyl group stabilises the carbocation intermediates.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
THRA 403 403Sidechain
ElectrostaticTransition state
Its hydroxyl group stabilises the carbocation intermediates.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ARGA 441 441Sidechain
ElectrostaticTransition state
It stabilises the additional negative charge on the departing diphosphate in the first step of the reaction.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 444 444Sidechain
Acid/baseResidue
It 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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
TYRA 520 520Sidechain
Acid/baseSubstrate
It donates a proton, accepted from Asp 525, to germacrene C6 in the formation of the eudesmane carbocation intermediate.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 525 525Sidechain
Acid/baseSubstrate
Acid/baseResidue
It 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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
TYRA 527 527Sidechain
ElectrostaticTransition state
The quadrupole of Tyr 527 stabilises the positive charge on C11, created after the formation of the C1-C10 bond.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9295271 Current protein Residue is positioned appropriately (ligand position known)
References:
1
Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase.
C. M. Starks and K. Back and J. Chappell and J. P. Noel
Science 277, (5333) 1815-20, (1997).
9295271
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