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

Abstract

Most 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.

A Single Active-Site Mutagenesis Confers Enhanced Activity and/or Changed Product Distribution to a Pentalenene Synthase from Streptomyces sp. PSKA01

“…The T182C variant exhibited both decreased total relative activity by 47.7% and an altered product distribution. Previous studies demonstrated that the pentalenene synthase mutants had other coproducts, such as germacrene A, protoilludene, α-humulene, and β-caryophyllene, besides the main product pentalenene [ 11 , 12 , 13 ]. Herein, the presence of product 6 is consistent with the previous report, while other products ( 2 , 3 , 4 , and 5 ) were discovered in this study for the first time.…”

“…Multiple-sequence alignment was generated by CLUSTALW and ESPript 3.0 [ 19 ]. The crystal structure of PenA (PDB: 6wkd) [ 12 ], which shares 92.9% of its sequence identity with PentS, was selected as a modeling template for the 3D structural analysis of PentS. A > 10 ns position-restrained MD simulation was performed to eliminate the steric conflicts of the modeled protein.…”

“…The F76W mutant produced a small amount of another anti-Markovnikov addition product, α-humulene, in addition to pentalenene, indicating that the benzene ring and pyrrole ring of tryptophan may stabilize the carbonium ion at C9 and C10. The F76H mutant generated trace α-humulene and a significant amount of Markovnikov addition product germacrene A, which was due to the resculpting of the active site such that a different conformation of farnesyl pyrophosphate (FPP) was stabilized in the binding cavity [ 12 ]. In addition, the only obvious basic residue in the active cavity is H309, which appears to be the ideal site for the deprotonation of carbocation intermediates to produce pentalenene.…”

Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches

“…Phe76, positioned in aforementioned region 1 in pentalenene synthase is involved in stabilisation of a positive charge which supports subsequent cyclisation involving less stable secondary carbocations rather than the much more stable tertiary carbocations stabilised in most other STSs. An aromatic residue at this position has been implicated in a similar role in other bacterial STSs involving secondary carbocations [65] . The I66F mutation in the diterpene synthase polytrichastrene synthase resulted in a major functional shift and the accumulation of several new products not produced by the native enzyme [39b]…”

“…An aromatic residue at this position has been implicated in a similar role in other bacterial STSs involving secondary carbocations. [65] The I66F mutation in the diterpene synthase polytrichastrene synthase resulted in a major functional shift and the accumulation of several new products not produced by the native enzyme. [39b] More systematic mutagenesis approaches yielded insights into the mutability landscape of TSs.…”