Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Abstract

Accession CodesThe atomic coordinates and structure factors for the DFFPP-PS complex have been deposited in the Protein Data Bank. RCSB PDB entry 6WKC (WT-Mg), 6WKD (WT-DFFPP), 6WKE DFFPP) for the respective models. ABBREVIATIONS FPP, farnesyl diphosphate; DFFPP, 12,13-difluorofarnesyl diphosphate; PS, pentalenene synthase, CS, caryolan-1-ol synthase. SUPPORTING INFORMATION AVAILABLE AbstractMost terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated.In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 Å above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-p interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76.Mutagenesis experiments also support a role for residue 76 in cation-p interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

Mechanistic Investigations on Microbial Type I Terpene Synthases through Site-Directed Mutagenesis

“…In pentalenene synthase, H309, adjacent to W308, protrudes into the active site 50 and was suspected to act as the catalytic base in the deprotonation of A1 to 3 (Scheme 2 ). 19 Following this hypothesis, in a next step, the same proton would be re-introduced to form A2 for the downstream steps towards 1 (as discussed above, recent investigations 37 favour a direct proton transfer from A1 to A2 mediated by F76). However, SDM yielding the enzyme variants H309A, H309S, H309C and H309F revealed a substantially retained enzyme activity in all cases, with strongest decreases for the H309F variant.…”

“…With the F76W mutant a mixture of 1 and α-humulene ( 3 ) was obtained, while F76H gave a partial functional switch, resulting in a mixture of the anti-Markovnikov products 1 and 3 , besides the Markovnikov adduct ( S )- 2 . 37…”