Probing Eudesmane Cation鈭捪€ Interactions in Catalysis by Aristolochene Synthase with Non-canonical Amino Acids

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• 作者：Juan A. Faraldos ; Alicja K. Antonczak ; Ver贸nica Gonz谩lez ; Rebecca Fullerton ; Eric M. Tippmann ; Rudolf K. Allemann
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：September 7, 2011
• 年：2011
• 卷：133
• 期：35
• 页码：13906-13909
• 全文大小：746K
• 年卷期：v.133,no.35(September 7, 2011)
• ISSN：1520-5126

文摘

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.