724783-68-0Relevant articles and documents
Aristolochene synthase: Mechanistic analysis of active site residues by site-directed mutagenesis
Felicetti, Brunella,Cane, David E.
, p. 7212 - 7221 (2004)
Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg2+-binding domains led in most cases to reductions in both kcat and kcat/K m as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in kcat that was offset by a 50-fold decrease in Km, resulting in a relatively minor 2-fold decrease in catalytic efficiency, kcat/Km. The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4).
Stabilisation of transition states prior to and following eudesmane cation in aristolochene synthase
Forcat, Silvia,Allemann, Rudolf K.
, p. 2563 - 2567 (2008/09/21)
The mechanistic details of the cyclisation of farnesylpyrophosphate (FPP) by aristolochene synthase (AS) from Penicillium roqueforti have only recently begun to emerge, mainly through the analysis of the reaction products generated by AS-mutants. The reaction proceeds through several intermediates including germacrene A and eudesmane cation. Previous work suggested that the side chain of phenylalanine 178 promoted the conversion of eudesmane cation to aristolochene. We now report that the catalytic function of this residue during the conversion of eudesmane cation to aristolochene is mainly due to the large size of its side chain, which facilitates the hydride shift from C2 to C3, rather than its aromatic character. In addition, F178 appears to control the regioselectivity of the final deprotonation step and, together with F112, helps stabilise the developing positive charge on C1 after the expulsion of pyrophosphate from the substrate. These results complete a screen of likely active-site aromatic residues and establish their respective roles in the conversion of FPP to aristolochene. The Royal Society of Chemistry 2006.
Germacrene A is a product of the aristolochene synthase-mediated conversion of farnesylpyrophosphate to aristolochene
Calvert, Melanie J.,Ashton, Peter R.,Allemann, Rudolf K.
, p. 11636 - 11641 (2007/10/03)
The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A. However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate. We demonstrate here for the first time that significant amounts of germacrene A (7.5% of the total amount of products) are indeed released from wild-type aristolochene synthase (AS) from Penicillium roqueforti. Germacrene A was identified through direct GC-MS comparison to an authentic sample and through production of β-elemene in a thermal Cope rearrangement. AS also produced a small amount of valencene through deprotonation of C6 rather than C8 in the final step of the reaction. On the basis of the X-ray structure of AS, Tyr 92 was postulated to be the active-site acid responsible for protonation of germacrene A (Caruthers, J. M.; Kang, I.; Rynkiewicz, M. J.; Cane, D. E.; Christianson, D. W. J. Biol. Chem. 2000, 275, 25533-25539). The CD spectra of a mutant protein, ASY92F, in which Tyr 92 was replaced by Phe, and of AS were very similar. ASY92F was approximately 0.1% as active as nonmutated recombinant AS. The steady-state kinetic parameters were measured as 0.138 min-1 and 0.189 mM for kcat and KM, respectively. Similar to a mutant protein of 5-epiaristolochene (Rising, K. A.; Starks, C. M.; Noel, J. P.; Chappell, J. J. Am. Chem. Soc. 2000, 122, 1861-1866), the mutant released significant amounts of germacrene A (～29%). ASY92F also produced various amounts of a further five hydrocarbons of molecular weight 204, valencene, β-(E)-farnesene, α- and β-selinene, and selina-4, 11-diene.
