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Catalytic Site Atlas

CSA LITERATURE entry for 1di1

E.C. namearistolochene synthase
SpeciesPenicillium roqueforti (Blue cheese mold)
E.C. Number (IntEnz) 4.2.3.9
CSA Homologues of 1di1There are 13 Homologs
CSA Entries With UniProtID Q03471
CSA Entries With EC Number 4.2.3.9
PDBe Entry 1di1
PDBSum Entry 1di1
MACiE Entry M0261

Literature Report

IntroductionAristolochene synthase is a fungal cyclase that catalyses the divalent metal dependent cyclisation of farnesyl diphosphate to the eremophilane sesquiterpene hydrocarbon aristolochene. Based on structural comparison, aristolochene is considered the likely parent hydrocarbon of several eremophilene toxins and bioregulators produced by a variety of filamentous fungi.
MechansimThe cyclisation mechanism is thought to be initiated by the ionisation of the allylic diphosphate ester to the corresponding allylic cation-pyrophosphate ion pair, followed by electrophilic attack at the C10 of the distal double bond and removal of a proton form the cis-C-12 methyl group, resulting in the formation of the intermediate (S)-geracrene A.
This intermediate undergoes further cyclisation by protonation at C-6 and intramolecular attack at the resultant carbocation to form the eudesmane cation. Successive 1,2 methyl migration and hydride shift followed by stereospecific deprotonation of H-8si leads to the formation of aristolochene. The observed sequence of anti-migration and syn-deprotonation takes place in a chair-boat conformation.
The tight control of the substrate conformation within the enzyme active site is thought to be the main contributing factor to the enzyme's activity. The divalent metal ions (not present in the pdb 1di1) trigger the initial carbocation formation by coordinating the the diphosphate leaving group.
Reaction

Catalytic Sites for 1di1

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
LysA206206macie:sideChainThe residue is thought to act as a general acid and general base within the cyclisation cascade. Hydrogen bonding interactions suggest Lys206 may be involved in a proton relay from the solvent, mediated by Arg200 and Asp203. Previous structural analysis implicated Tyr72 at the general acid/base catalyst. However, mutagenesis studies have shown the Tyr72Phe mutant to still product aristolochene as the major product (>81%).
PheA112112macie:sideChain The aromatic side chain is positioned to stabilise the developing partial positive charge on C-1 in the first step of catalysis, as well as enforcing the substrate into a reactive conformation and controlling the reaction stereochemistry.
PheA178178macie:sideChainThe residue's aromatic side chain is correctly positioned to stabilise the developing positive charge on C-1 in the first step of catalysis. This electrostatic stabilisation of high energy intermediates is achieved without the risk to quenching the carbocation and annihilating catalytic activity.
TrpA333334macie:sideChainThe residue is positioned to stabilise the developing positive charge on C-3 in the eudesmane cation intermediate.

Literature References

Notes:There is some debate over the role of Tyr72 and Lys206.
Miller DJ
Stereochemistry of eudesmane cation formation during catalysis by aristolochene synthase from Penicillium roqueforti.
Org Biomol Chem 2008 6 2346-2354
PubMed: 18563268
Felicetti B
Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis.
J Am Chem Soc 2004 126 7212-7221
PubMed: 15186158
Caruthers JM
Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti.
J Biol Chem 2000 275 25533-25539
PubMed: 10825154
Miller DJ
6- and 14-Fluoro farnesyl diphosphate: mechanistic probes for the reaction catalysed by aristolochene synthase.
Org Biomol Chem 2009 7 962-975
PubMed: 19225680
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