Enzyme Access Tunnel Engineering in Baeyer-Villiger Monooxygenases to Improve Oxidative Stability and Biocatalyst Performance

Hydrogen peroxide is involved in a variety of enzyme catalysis as an oxidant or toxic by-product. Thereby, attenuation of the H₂O₂-driven oxidative stress is one of the key issues for preparative biocatalysis. Here, a rational approach to improve the robustness of enzymes, in particular, Baeyer-Villiger monooxygenases (BVMOs) against H₂O₂ was investigated. The enzyme access tunnels, which may serve as exit paths for H₂O₂ from the active site to the bulk, were predicted by using the CAVER and/or protein energy landscape exploration (PELE) software for the phenylacetone monooxygenase variant (PAMO_C65D) from Thermobifida fusca and the BVMO from Pseudomonas putida KT2440. The amino acid residues, which are susceptible to oxidation by H₂O₂ (e. g., methionine and tyrosine) and located in vicinity of the predicted H₂O₂ migration paths, were substituted with less reactive or inert amino acids (e. g., leucine and isoleucine). This led to design of the H₂O₂-resistant enzyme variants, which became robust biocatalysts for synthetic applications. For instance, the H₂O₂-resistant P. putida BVMO reached turnover numbers of 4,100 for the BV oxygenation of 4-decanone, which is 2.8-fold greater than the parent enzyme. Moreover, the H₂O₂-resistant P. putida BVMO allowed 2-fold enhancement in titer of 9-(nonanoyloxy)nonanoic acid (8) formation in a cascade fatty acid biotransformation. Therefore, it was assumed that the CAVER/PELE-based H₂O₂ migration path engineering represents an efficient rational design approach to improve not only oxidative stability but also biotransformation performance of the H₂O₂-forming or utilizing enzymes (e. g., BVMOs, oxidases, and peroxidases). (Figure presented.).