Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Abstract

Baeyer-Villiger monooxygenase (BVMO)-mediated regiodivergent conversions of asymmetric ketones can lead to the formation of "normal" or "abnormal" lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active-site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2-oxo-Δ -4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase from Pseudomonas putida (OTEMO) for cis-bicyclo[3.2.0]hept-2-en-6-one (1) and trans-dihydrocarvone (2), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild-type enzyme converted (-)-1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)-2, whereas the wild-type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO and CHMO ) to switch their regioselectivity towards (+)-2, which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.

Biocatalytic Routes to Lactone Monomers for Polymer Production

“…This suggests the WT selectivity (modeled by M(med_W)) arises from a clear thermodynamic preference for the eq, eq conformer throughout, leading to the The mutations were initially identified by Balke et al 79 after docking of the normal lactone product of (2R,5R)-(+)-dihydrocarvone (ax, ax substrate conformer) into protein crystal structure geometries of the WT; in later work, a different lactone product, from the reaction of the (2S, 5S)-(+)dihydrocarvone enantiomer, was also considered. 57 These considerations led the authors to propose the sites for mutation, aimed at opening up the active site to better accommodate the normal lactone, with experimental evaluation used to select the final triple site mutation (F249A, F280A, F435A in the organism used here). As shown in Figure 2A for the relevant Criegee intermediates, these mutations do indeed give rise to a comparatively more open site.…”

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

“…However, mutation studies with the corresponding residues in CHMO, PAMO (W501) and 2-oxo-∆ 3 -4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase (OTEMO, W501) have revealed that it contributes to the regioselectivity of the enzymes. In OTEMO, W501A/V shifted the conversion of (-)-cis-bicyclo[3.2.0]hept-2-en-6-one from equimolar products to heavily favour the distal lactone (95%) [31]. In PAMO, an inverse in the ratio of normal (proximal) to abnormal (distal) lactone from rac-cis-bicyclo[3.2.0]hept-2-en-6-one was observed [28].…”

Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability

“…Type I is tightly bound to flavin adenine dinucleotide (FAD) as a cofactor and uses reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a source of electrons, whereas type II relies on flavin mononucleotide (FMN) and uses reduced nicotinamide adenine dinucleotide (NADH) as an electron donor . Type I BVMOs catalyze the oxidation of ketones into esters or lactones, with exceptionally high regio‐, chemo‐, and enantioselectivity for the production of fine chemicals or chiral building blocks . Based on these features, many industrial applications have been suggested, but, due to low operational stability under given reaction conditions, exploitation on a large scale is still challenging .…”