Biocatalytic Baeyer–Villiger Reactions: Uncovering the Source of Regioselectivity at Each Evolutionary Stage of a Mutant with Scrutiny of Fleeting Chiral Intermediates

Abstract

In this study, we report the discovery of unexpected mechanistic intricacies of Baeyer–Villiger monooxygenases (BVMOs) and provide insights that promise to help in extending their applications in synthetic organic chemistry and biotechnology. The basic mechanism of BVMOs as catalysts in the oxidation of unsymmetrical ketones R1–(CO)–R2 is well known, which involves the intermediacy of short-lived Criegee intermediates. The tendency of R1 or R2 to migrate preferentially in the breakdown of the Criegee intermediate follows the traditional requirement of an antiperiplanar conformation with maximum stabilization of the incipient positive charge. The challenge of inverting the regioselectivity of group migration with the formation of abnormal products was recently met by the semi-rational directed evolution of TmCHMO with the generation of a quadruple mutant. Although a reasonable model explaining the mutational effect was suggested, the theoretical analysis did not include the calculation of both enantiomeric forms of the fleeting chiral Criegee intermediate in transition states and focused only on the wild-type enzyme and the quadruple mutant. The present investigation utilizes complete mutational deconvolution with the experimental construction of a fitness-pathway landscape comprising 4! = 24 upward climbs. We were confronted by the discovery that the absolute configuration of the Criegee intermediate switches from (R) to (S), depending upon the stage of the evolutionary process. On the basis of X-ray structural data, the physical basis of this phenomenon was illuminated by quantum chemical analyses performed on the enzymes at all evolutionary steps of a selected pathway. The hitherto unexplored role of fleeting chiral intermediates in the mechanism of other enzyme types deserves increased attention.

Redesigning an (R)-Selective Transaminase for the Efficient Synthesis of PharmaceuticalN-Heterocyclic Amines

“…To decrease the screening effort, a “simplified amino acid alphabet” was introduced. This group consisted of amino acids of different sizes and polarity/hydrophobicity to fine-tune the binding pocket . Each of the selected target residues was substituted by amino acids in the reduced amino acid alphabet (namely, Phe, Asn, Val, or Ala) to construct a small but smart variant library.…”

“…Several successful examples of using reduced amino acid alphabets in enzyme engineering have been reported. For example, a twelve-amino-acid alphabet (Phe, Leu, Ile, Val, Tyr, His, Asn, Asp, Cys, Arg, Ser, and Gly) and a seven-amino-acid alphabet (Ala, Cys, Ile, Leu, Ser, Thr, and Val) were used to construct small but smart libraries with a high frequency of enhanced variants for epoxide hydrolase. , Similarly, a simplified four-amino-acid alphabet (Phe, Asn, Val, and Ala) was designed to regulate epoxide hydrolase regioselectivity …”

“…Therefore, to construct a smaller but higher-quality variant library, we used the “reduced amino acid alphabet” technique. This method involves selecting degenerate codons that encode a small number of amino acids . Several successful examples of using reduced amino acid alphabets in enzyme engineering have been reported.…”

Redesigning Enzymes for Biocatalysis: Exploiting Structural Understanding for Improved Selectivity

“…These techniques are widely used to enable the interpretation of experimental data to understand enzymatic mechanisms and rationalize the enzyme selectivity in several of the enzyme families discussed in this review including ene-reductases (Lonsdale and Reetz, 2015), diels-alderase (Byrne et al, 2016), BVMOs regioselectivity (Li G. et al, 2018) (Dong et al, 2022) and ketoreductase stereoselectivity (Serapian and Van Der Kamp, 2019).…”

Engineered P450 Atom-Transfer Radical Cyclases are Bifunctional Biocatalysts: Reaction Mechanism and Origin of Enantioselectivity

“…Such intuitive substrate binding models obtained from experimental X-ray structures and computational substrate docking and/or classical molecular dynamics (MD) simulations have been widely used in biocatalysis and protein engineering. Nevertheless, an increasing number of studies underscored the importance of interrogating transition-state models to gain an accurate understanding of enzymatic stereoselectivities, , especially when the reactive functional group of the substrate (e.g., an olefin) does not strongly interact with the protein scaffold and is flexible in the enzyme–substrate complex (Figure d). In this situation, substrate binding models become ineffective, and computational models based on transition-state analysis are critical to describe the origin of enzymatic stereocontrol.…”