title: Probing eudesmane cation-π interactions in catalysis by aristolochene synthase with non-canonical amino acids.
abstract: 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.
authors.affiliation: School of Chemistry, Cardiff University, Park Place, Cardiff, United Kingdom.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Faraldos
authors.forename: Alicja K
authors.initials: AK
authors.lastname: Antonczak
authors.forename: Verónica
authors.initials: V
authors.lastname: González
authors.forename: Rebecca
authors.initials: R
authors.lastname: Fullerton
authors.forename: Eric M
authors.initials: EM
authors.lastname: Tippmann
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D012717:Sesquiterpenes / Q000378:metabolism; D045787:Sesquiterpenes, Eudesmane / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002412:Cations; D012717:Sesquiterpenes; D045787:Sesquiterpenes, Eudesmane; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; C015872:eudesmane; D007535:Isomerases; C060050:aristolochene synthase