The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

Abstract

Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution.Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9en-7-ol. The 11-step in-enzyme carbocation cascade is compared with the same reaction in the absence of the enzyme. Remarkably, the free energy profiles in gas phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π−cation, dipole−cation, and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design.

Redesigning the Molecular Choreography to Prevent Hydroxylation in Germacradien-11-ol Synthase Catalysis

“…36 In the hydroxylating diterpene synthase CotB2, extensive experimental and computational work has highlighted key active site interactions and suggestions of how these are altered by mutations that lead to different main products. 16,37 However, a complete picture of how (sesqui)terpene synthases mediate water capture remains elusive.…”

“…Terpenoids are the largest group of natural products universally present in all living systems with immense diversity in their structure and functions. , To date, over 80000 naturally occurring isoprenoids have been reported, including more than 7000 sesquiterpenoids derived from ∼300 stereochemically distinct hydrocarbon skeletons. , A highly complex and structurally diverse array of hydrocarbon or hydroxylated terpenes is synthesized by terpene synthase catalysis from acyclic isoprenyl diphosphates such as geranyl diphosphate (GDP), farnesyl diphosphate (FDP), and geranylgeranyl diphosphate (GGDP). , The diversity of terpene skeletons arises not only from the number of terpene synthases but also from the ability of some terpene synthases to form multiple products from a single substrate. − The product specificity and activity of terpene synthases are highly dependent on a small number of amino acids present inside or near the hydrophobic active site pocket. Previous studies have shown the profound effect that slight changes to these amino acids can have on the electronic distribution and the geometry of the active site pocket, which in turn affect the enzyme activity and specificity. − …”

“…Mutation analysis of hedycaryol synthase showed the catalytic involvement of Asp82 of the DDXXD motif in the activation of water for the hydroxylation, whereas in bacterial cineole synthase, a specific active site Asn has been implicated in water activation . In the hydroxylating diterpene synthase CotB2, extensive experimental and computational work has highlighted key active site interactions and suggestions of how these are altered by mutations that lead to different main products. , However, a complete picture of how (sesqui)­terpene synthases mediate water capture remains elusive.…”

1,2‐ or 1,3‐Hydride Shifts: What Controls Guaiane Biosynthesis?

“…These transformations involve just a single enzyme catalyzed reaction and proceed through cationic cascade reactions inside a hydrophobic cavity of the TPS. Because of their transient nature the cationic intermediates along the cascade cannot be observed spectroscopically, but especially isotopic labeling experiments[ 6 , 7 ] and DFT or QM/MM calculations[ 8 , 9 , 10 , 11 , 12 , 13 ] have helped to develop a deep mechanistic understanding of TPS catalysis. Also structure based site‐directed mutagenesis can give valuable insights,[ 14 , 15 , 16 ] especially if an enzyme variant leads to an aberrant product formed by deprotonation of a cationic intermediate, giving indirect evidence for its existence.…”

Structural and Molecular Basis of the Catalytic Mechanism of Geranyl Pyrophosphate C6‐Methyltransferase: Creation of an Unprecedented Farnesyl Pyrophosphate C6‐Methyltransferase

“…However,our QM/MM analysis estimated the contribution of the cation-p interaction to the stabilization of TS1 to be only 1.2 kcal mol À1 .B ecause the QM/MM calculation indicated that the carbocation formed in the intermediate state is atertiary cation at C7 (Figure S12), the carbocation should be relatively stable and the effect of cation-p stabilization caused by F210 should be minor.Generally,aromatic residues located in the active site pockets of enzymes (e.g., terpene cyclase [1] )are proposed to play an important role in stabilizing carbocation intermediates by cation-p interactions.However, our results indicated that steric hindrance caused by such aromatic residues is more important to stabilize the intermediate in some cases.Recently,DFT and QM/MM analyses of the diterpene cyclase CotB2, which catalyzes the formation of cyclooctat-9-en-7-ol from GGPP,r evealed that the free energy profiles of the reaction in CotB2 were very similar to those in the gas phase. [27] Therefore,c ation-p interactions may not necessarily be involved in the enzymatic reactions catalyzed by terpene cyclasesa nd methyltransferases.…”