Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Abstract

The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer-Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer-Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to "designed" directed evolution that address the binding site directly.allosteric effects | enzymes | molecular dynamics simulations | protein engineering P rotein allostery has been recognized as a positive or negative cooperative event, leading to a structural change at the binding site as a result of distal docking of a molecule acting as an effector (1-5). Allosteric effects can be influenced by such factors as variation in pH, temperature, ionic strength, and covalent modification as well as mutational changes. A variety of experimental and computational techniques have been utilized to unravel the intricacies of this phenomenon (1-5), but also to achieve useful applications such as the creation of protein switches (6). Among the techniques that have been applied to address these challenges is directed evolution, a method that is generally used to engineer the catalytic profiles of enzymes or the binding properties of proteins (7-11). In the endeavor to make directed evolution of enantioselective enzymes more efficient than in the past, we recently introduced the concept of iterative saturation mutagenesis according to which sites around the binding pocket are randomized iteratively, a given site being composed of one or more amino acid positions in the protein (12-14.) When applying this technique to enzymes displaying strong allosteric effects or coupled motions along the reaction coordinate (15), it is necessary to consider such phenomena to make reasonable...

Exploring the Structural Basis of Substrate Preferences in Baeyer-Villiger Monooxygenases

“…In other words, there is substrate selectivity and preference, with, however, a considerable level of promiscuity as effectively indicated by the acceptance of both progesterone and phenylacetone by STMO. A key point that will be subject to further studies is the dynamics and flexibility of the active site, especially with regard to the role of conformational adaptability (26). The substrate preference is apparently the result of the balance of relatively nonspecific, mostly hydrophobic, interactions that the groups on the surface of the binding site are able to establish with the substrate ligands.…”

Snapshots of Enzymatic Baeyer-Villiger Catalysis

“…The three-dimensional structure of PAMO has been solved revealing a two-domain organization (Fig. 1A) (16), and its catalytic cycle has been subject of extensive kinetic studies (17)(18)(19)(20)(21)(22). More recently, the structure of cyclohexanone monooxygenase in complex with NADP ϩ highlighted a proposed mechanism for the dual catalytic role of NADP(H) (9,23).…”

Evolutionary Engineering of Artificial Proteins with Limited Sets of Primitive Amino Acids

“…The NDT library produced many more variants with high activity than the NNK library (Reetz et al, 2008). Moreover, the authors succeeded in modifying other enzymes using a library based on the randomized codon NDT, for example, by inducing allosteric effects into Baeyer-Villiger monooxygenase (Wu et al, 2010) and manipulating the stereoselectivity of limonene epoxide hydrolase (Zheng & Reetz, 2010). Fellouse et al (2004Fellouse et al ( , 2005 demonstrated that the performance of a randomized antibody library was maintained when the number of amino acid types constituting part of a randomized complementarity-determining region (CDR) in the library was reduced to just four (Ala, Ser, Asn and Tyr) or even two (Ser and Tyr).…”

“…Although protein engineering using a limited set of primitive amino acids might improve protein folding ability and the frequency of occurrence of functional proteins, the need remains to determine the most appropriate subset of amino acids for functional selection because our study (Tanaka et al, 2011) and those of Reetz's group (Reetz et al, 2008;Wu et al, 2010;Zheng & Reetz, 2010) and Fellouse et al (2004Fellouse et al ( , 2005) simultaneously compared only a few subsets of amino acids. Some putative new amino acids may be essential for some structures and functions.…”

“…Recently, it has been reported that such limited sets of amino acids are effective for functional selection from randomized libraries (Reetz et al, 2008;Wu et al, 2010;Zheng & Reetz, 2010), although only a few amino acids were randomized in the active sites in these studies. Reetz et al compared the quality of randomized libraries in which five amino acid residues around the active site of epoxide hydrolase were replaced by 20 kinds of amino acids encoded by NNK (K = T or G) or 12 kinds of amino acids (Gly, Asp, Val, Ser, Leu, Arg, Asn, Ile, His, Cys, Phe and Tyr) encoded by NDT (D = T, A or G).…”