Laboratory Evolution of Robust and Enantioselective Baeyer−Villiger Monooxygenases for Asymmetric Catalysis

Abstract

The Baeyer-Villiger Monooxygenase, Phenylacetone Monooxygenase (PAMO), recently discovered by Fraaije, Janssen, and co-workers, is unusually thermostable, which makes it a promising candidate for catalyzing enantioselective Baeyer-Villiger reactions in organic chemistry. Unfortunately, however, its substrate scope is very limited, reasonable reaction rates being observed essentially only with phenylacetone and similar linear phenyl-substituted analogs. Previous protein engineering attempts to broaden the range of substrate acceptance and to control enantioselectivity have been met with limited success, including rational design and directed evolution based on saturation mutagenesis with formation of focused mutant libraries, which may have to do with complex domain movements. In the present study, a new approach to laboratory evolution is described which has led to mutants showing unusually high activity and enantioselectivity in the oxidative kinetic resolution of a variety of 2-aryl and 2-alkylcyclohexanones which are not accepted by the wild-type (WT) PAMO and of a structurally very different bicyclic ketone. The new strategy exploits bioinformatics data derived from sequence alignment of eight different Baeyer-Villiger Monooxygenases, which in conjunction with the known X-ray structure of PAMO and induced fit docking suggests potential randomization sites, different from all previous approaches to focused library generation. Sites harboring highly conserved proline in a loop of the WT are targeted. The most active and enantioselective mutants retain the high thermostability of the parent WT PAMO. The success of the "proline" hypothesis in the present system calls for further testing in future laboratory evolution studies.

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ 3 -4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

“…In this scenario, the formation and disruption of hydrogen-bonding interactions between two main-chain carbonyl groups in this region (T442 and C444) and the critical R337 in the open and closed forms, respectively, may play an important role for this purpose. The effects of residues 440 to 446 in PAMO (same numbering in OTEMO) have been extensively studied, and an important role of this segment in conferring substrate specificity and enantioselectivity has been well established (46,47). In addition, a very recent report also identified A435 as contributing to substrate specificity (19).…”

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

“…Furthermore, the combination of structural and enzyme engineering studies has provided important hints about the protein elements that determine the substrate selectivity of the enzymes. For instance, a loop in direct contact with the pyrimidine ring of the flavin has been shown to be crucial for substrate binding (15). On the other hand, especially with the aim of exploiting these enzymes for biocatalytic purposes, we need to further understand and rationalize their substrate specificity properties.…”

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).…”