Aristolochene Synthase: Mechanistic Analysis of Active Site Residues by Site-Directed Mutagenesis

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Abstract
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/Km 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).