Substrate Pre‐Folding and Water Molecule Organization Matters for Terpene Cyclase Catalyzed Conversion of Unnatural Substrates

Abstract

Terpene cyclase enzymes have recently been challenged with terpene substrate derivatives to generate additional chemical complexity beyond to what is currently found in nature. Herein, molecular dynamics and biocatalysis are used to shed light on the flexibility and inherent limitation of a triterpene cyclase in converting unnatural substrates. Our studies suggest that populating binding modes which allows for concerted reaction pathways is a key element towards an expanded substrate scope and new chemistries displayed by terpene cyclases. Additionally, we show that the spatial organization of water, which is influenced by both the substrate architecture as well as the active site geometry, controls the product selectivity. This highlights that activity and selectivity displayed by terpene cyclases acting on unnatural substrates is particularly difficult to predict, since they depend on various parameters.

Protonation‐Initiated Cyclization by a Class II Terpene Cyclase Assisted by Tunneling

“…In analogy, a protonation distance of 3.2 Å for the corresponding heavy atoms has been suggested based on analysis by AutoDock . Molecular dynamics (MD) simulations of the triterpene cyclase from Alicyclobacillus acidocaldarius , which also operates by the class II mechanism and displays the same fold as that of PtmT2 (Figure A), have shown that very short protonation distances down to 1.7 Å are feasible . Compressed internuclear distances have been attributed to “tunnel‐ready” conformations …”

Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants

“…In contrast to the cyclization of farnesol, the incubation of geraniol with purified recombinant Aac SHC wild type afforded a very small amount of its cyclization product. This may reflect the fact that the active site of the enzyme does not favour reactive binding of the geraniol substrate . Our previous studies suggested that alteration of the active site pocket of Aac SHC facilitated the conversion of truncated terpene‐like substrates without disturbing the important catalytic machinery …”

“…Starting from small sesquiterpenes such as farnesol (C 15 ), up to C 35 unnatural polyprenoids can be successfully converted . SHCs can also be used for carbon–carbon, carbon–oxygen, and carbon–nitrogen bond‐formation reactions by using functionalities such as alcohols, aromatic systems, carboxylic acids, ketones, and amides . Previous work demonstrated that SHCs could perform Friedel–Crafts alkylations with aromatics in addition to hydroamidations, which revealed the potential of these enzymes in chiral Brønsted acid catalysis …”

“…Recent investigations of Syrén et al. on the cyclization of geraniol and geranyl alkyl ethers by Aac SHC revealed that increased alkyl substituents provided increased activities for the cyclization and increased selectivities towards the geranyl alkyl ether …”

Selectivity in the Cyclization of Citronellal Introduced by Squalene Hopene Cyclase Variants

“…These synthetic hosts contain well‐defined cavities for stabilization of high‐energy intermediates and transition states. The proposed mechanism of catalysis is similar to that employed by terpene cyclase enzymes, which control the reaction outcomes by selectively stabilizing cationic intermediates and transition structures through multiple interactions with aromatic residues lining the active site …”