Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer–Villiger Monooxygenase

Abstract

The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer–Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C T m ) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.

A Cold-Active Flavin-Dependent Monooxygenase from Janthinobacterium svalbardensis Unlocks Applications of Baeyer–Villiger Monooxygenases at Low Temperature

“…Nevertheless, there is currently no rationale known for increasing enzyme activity at low temperatures. Therefore we believe that JsFMO represents an excellent basis for further engineering studies. , …”

“…Therefore we believe that JsFMO represents an excellent basis for further engineering studies. 41 , 42 …”

“…Therefore we believe that JsFMO represents an excellent basis for further engineering studies. 41,42 The active site of cold-active enzymes is particularly flexible compared to the rest of the structure. Consequently, they are and BVMO-I.…”

The Al-Containing Silicates Modified with Organic Ligands and SnO2 Nanoparticles for Catalytic Baeyer-Villiger Oxidation and Aerobic Carboxylation of Carbonyl Compounds

“…The high cost and non-recyclability of catalysts also retarded their wide application [ 10 , 11 ]. On the other hand, biocatalysts such as enzyme-based substances were explored for BVO of ketone in order to avoid metal pollution [ 12 ]. In practice, high chemoselectivity (migratory aptitude) could be obtained, but there were still problems with the availability, recovery and cost of the catalyst, along with product purification [ 12 ].…”

Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media

“…Among various biotransformations, the oxygenation reactions inter‐cooperating molecular oxygen (O 2 ) to generate value‐added chemicals have been of great interest in the biocatalysis community [7–9] . In this context, Baeyer‐Villiger monooxygenases (BVMOs) are known to catalyze the regio‐ and enantioselective Baeyer‐Villiger oxidations and sulfoxidations for the synthesis of a wide range of products, including bioactive compounds or chiral precursors [10–14] . Despite the extensive exploration of BVMOs, there are still several limitations linked to the practical use of BVMOs in aqueous media including i) enzyme instability, ii) nicotinamide cofactor dependence, iii) product auto‐hydrolysis, iv) relatively low oxygen solubility, and v) the substrate and product inhibition present for most BVMOs [15] .…”