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Probing Eudesmane Cation−π Interactions in Catalysis by Aristolochene Synthase with Non-canonical Amino Acids

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journal contribution
posted on 2011-09-07, 00:00 authored by Juan A. Faraldos, Alicja K. Antonczak, Verónica González, Rebecca Fullerton, Eric M. Tippmann, Rudolf K. Allemann
Stabilization of the reaction intermediate eudesmane cation (3) through interaction with Trp 334 during catalysis by aristolochene synthase from Penicillium roqueforti was investigated by site-directed incorporation of proteinogenic and non-canonical aromatic amino acids. The amount of germacrene A (2) generated by the mutant enzymes served as a measure of the stabilization of 3. 2 is a neutral intermediate, from which 3 is formed during PR-AS catalysis by protonation of the C6,C7 double bond. The replacement of Trp 334 with para-substituted phenylalanines of increasing electron-withdrawing properties led to a progressive accumulation of 2 that showed a good correlation with the interaction energies of simple cations such as Na+ with substituted benzenes. These results provide compelling evidence for the stabilizing role played by Trp 334 in aristolochene synthase catalysis for the energetically demanding transformation of 2 to 3.


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