Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

Abstract

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio-or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.Enzymes have been gaining increasing attention as efficient and selective catalysts to be used in synthetic chemistry. Baeyer-Villiger monooxygenases (BVMOs) comprise a group of enzymes that are particularly interesting for synthetic applications. These biocatalysts employ molecular oxygen as a mild oxidant to oxidize carbonylic compounds. Apart from catalyzing Baeyer-Villiger reactions, BVMOs are capable of oxidizing a range of heteroatoms (e.g., sulfur, nitrogen, and boron). Furthermore, they often perform these reactions with high chemo-, regio-, and enantioselectivity (5, 15, 30). The use of oxygen, which is a cheap and clean oxidant, and their diversity of catalyzed reactions make BVMOs attractive candidates for biocatalytic processes.The identification of phenylacetone monooxygenase (PAMO) in the moderately thermophilic bacterium Thermobifida fusca has brought about a breakthrough in the research on BVMOs (11). PAMO is a thermostable enzyme that can easily be expressed in Escherichia coli and purified. Besides, its crystal structure has been solved as the first structure of a BVMO (17). While PAMO shows excellent stability, even in the presence of organic solvents (6, 28), its substrate specificity is rather restricted. The enzyme accepts mainly small aromatic ketones and sulfides (7, 26), whereas the oxidations of bulkier ketones occur with lower activity and selectivity (27). However, the unique...

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

“…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). The OTEMO structures reported here reveal for the first time that this region could undergo significant conformational adjustments and provide detailed pictures of different substrate-binding environments.…”

Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites

“…Acetaminophen was purchased from Fluka (Buchs, Switzerland), ultra-pure high-pressure liquid chromatography (HPLC)-grade acetonitrile and HPLC grade methanol were purchased from Biosolve (Valkenswaard, the Netherlands). We purchased catalase from Fluka, and phosphite dehydrogenase was prepared using an established protocol (Dudek et al, 2011). Ultrapure water was obtained from a Milli-Q Advantage A10 Water Purification system (Millipore Corp., Billerica, MA).…”

Polycyclic Ketone Monooxygenase (PockeMO): A Robust Biocatalyst for the Synthesis of Optically Active Sulfoxides

“…Yet, all these BVMOs suffer from low operational stability. Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is one of the few known BVMOs that is stable at elevated temperatures [25,[30][31][32][33], and it tolerates, to some extent, the use of cosolvent; in fact, some cosolvents even improve its performance [28]. Nevertheless, all of the above-mentioned BVMOs have the drawback that their substrate profile is restricted to relatively small substrates.…”

“…HPLC complete data are supplied at the Supplementary Material. The sulfoxide configurations were established by comparing the HPLC chromatograms with the ones described in the bibliography [24,31,33].…”