Squalene hopene cyclases are protonases for stereoselective Brønsted acid catalysis

Abstract

For many important reactions catalyzed in chemical laboratories, the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Brønsted acid catalysis, a powerful method to promote a myriad of chemical transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymatic Brønsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chemistry. This work highlights the potential of systematic investigation on nature's catalytic machineries to generate unique catalysts.

Squalene-hopene cyclases—evolution, dynamics and catalytic scope

“…Active site mutations of AacSHC enable the subtly reshape of the binding pocket directing the SHC toward an artificial 'protonase' [45 ] able to perform novel C-C and C-X bond transformations. The fact that single amino acid exchanges could afford such great improvements in reactivity suggests that these catalysts on non-natural reactions will be evolvable.…”

“…Although AacSHC features prominently the activation and cyclization of (S)-citronellal (S)-22, an intensive study of the active site constitution of AacSHC revealed mutant combinations which inverted the natural substrate promiscuity from (S)-citronellal (S)-22 to the corresponding (R)-enantiomer (R)-22 yielding moderate to excellent selectivities of the corresponding isopuleogol isomers (S)-23, (S)-24, (R)-25 and (R)-26 (d.e. 16-99%) [45 ].…”

“…Rational libraries using aliphatic amino acids as substitution residues were previously used by the group of Hoshino for the generation of structurally diverse natural products, including novel triterpene skeletons (monocyclic to pentacyclic products) [44 ]. In this light, the design concept for fine-tuned and re-sized active sites of the AacSHC aimed for the conversion of truncated terpene-like substrates by substitution of the active site amino acid residues without disturbing the catalytically important Brønsted acid machinery [45 ]. The introduction of smaller (Gly and Ala) and of rigid aromatic residues (Phe and Trp), which would preserve the hydrophobic nature of the active site, was explored to enter novel routes, alternative to those defined by evolution, for binding of smaller terpenes and for activating of epoxides and carbonyl substrates.…”

Expanding the Enzyme Universe: Accessing Non‐Natural Reactions by Mechanism‐Guided Directed Evolution

“…In a very recent study, 59 Hauer and co-workers engineered an SHC from A. acidocaldarius ( Aac SHC) to improve the catalytic activity for several different modes of Brønsted acid-catalyzed cyclizations, including the Prins cyclization of ( S )-citronellal (Figure 13). Screening a library of enzyme variants made by mutating several amino acids in proximity to the catalytic Asp376 residue, they discovered variants with greatly improved activities for various cyclization reactions.…”

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

“…Hence, enzyme promiscuity is of immense relevance for the chemical and biotechnological industrial fields for which the development of hitherto unknown biochemical reactions is of central interest [8]. For instance, based on chemical expertise, researchers have successfully discovered novel chemistries for stereoselective carbon-carbon bond formation [14] in existing enzyme scaffolds that can be significantly improved using enzyme engineering [44]. An enhanced understanding of promiscuous activities and their evolution at the molecular level would thus facilitate the transition towards a more sustainable society for which chemical transformations could be replaced by green enzymatic reactions [4].…”