Daniel E. Torres Pazmiño

Baeyer–Villiger monooxygenases: recent advances and future challenges

Baeyer-Villiger monooxygenases For many enzyme classes, a wealth of information on, for example, structure and mechanism has been generated in the last few decades. While the first Baeyer-Villiger monooxygenases (BVMOs) were already isolated more than 30 years ago, detailed data on these enzymes were lacking until recently. Over the last years several major scientific breakthroughs, including the elucidation of BVMO crystal structures and the identification of numerous novel BVMOs, have boosted the research on BVMOs. This has led to intensified biocatalytic explorations of novel BVMOs and structure-inspired enzyme redesign. This review provides an overview on the recently gained knowledge on BVMOs and sketches the outlook for future industrial applications of these unique oxidative biocatalysts.

Self-Sufficient Baeyer–Villiger Monooxygenases: Effective Coenzyme Regeneration for Biooxygenation by Fusion Engineering†

Over the past few years, industrial interest in biocatalysts that perform selective oxidative reactions has increased significantly. Baeyer–Villiger monooxygenases (BVMOs) have been identified as a highly versatile class of enzymes for the efficient catalysis of chemo-, regio-, and/or enantioselective oxygenation reactions. Although the most prominent transformation catalyzed by these biocatalysts is a chiral variant of the classical Baeyer–Villiger reaction, the oxygenation of heteroatoms and epoxidation reactions have also been reported. Stoichiometric amounts of O2 and NADPH are required for these reactions. A complication for the largescale application of these reactions is the high cost of the reduced nicotinamide coenzyme. To overcome this problem, several electrochemical and photochemical approaches have been explored. However, the efficiency of these approaches is typically poor. Furthermore, it has been shown that BVMOs require NADP for stability and enantioselective catalysis. An efficient and commonly used method for coenzyme regeneration employs whole cells, especially in combination with the recombinant expression of the required biocatalysts. This strategy has been implemented in BVMOmediated biotransformations with wild-type strains and has proved particularly successful with recombinant overexpression systems. The approach avoids laborious enzyme purification steps and exploits the coenzyme regeneration capacity of the host. Although whole cells have been shown to be effective catalysts for Baeyer–Villiger oxidation, they also exhibit limitations, such as cellular toxicity, enzyme inhibition by the substrate/product, degradation of the product, and poor oxygen-transfer rates. Coenzyme regeneration by using isolated enzymes has also been studied extensively in the past few years. Well-known examples of such NADPH-regenerating enzymes are alcohol dehydrogenase and formate dehydrogenase. A phosphite dehydrogenase (PTDH) was also identified as an effective enzyme for coenzyme regeneration. The favorable thermodynamic equilibrium constant makes the oxidation of phosphite a nearly irreversible process. The exquisite selectivity of PTDH for phosphite also precludes any side reactions, such as those that can occur, for example, when an alcohol dehydrogenase is used. These characteristics make PTDH an ideal candidate for use as a coenzyme regenerating enzyme (CRE) in combination with BVMOs or other NAD(P)H-dependent enzymes. Herein, we report a novel approach to the combination of the catalytic activity of a redox biocatalyst with concomitant coenzyme recycling in a single fusion protein (Scheme 1). During the last decade, a number of fusion protein tags have been developed. These tags are used intensely in life-sciencerelated research and commercial activities. Although the fusion of proteins is a widely applied strategy in, for example, enzyme purification (e.g. the use of glutathione S transferase (GST) tags) and the subcellular visualization of target proteins (e.g. with a green fluorescent protein (GFP) tag), this concept is hardly ever encountered in the context of synthetic applications. Only a few isolated examples in the literature provide evidence that the fusion of separate enzymes can result in improved biocatalytic properties. We report herein on the engineering of a number of representative BVMOs that are linked covalently to soluble NADPH-regenerating phosphite dehydrogenase. This construct enables the use of phosphite as a cheap and sacrificial electron donor with whole cells, cell extracts, and purified enzyme. It was our particular goal to design a self-sufficient two-in-one redox biocatalyst that does not require an additional catalytic entity for coenzyme recycling. As model

Snapshots of Enzymatic Baeyer-Villiger Catalysis: Oxygen Activation and Intermediate Stabilization.

Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP+ ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and “Criegee-stabilizing” catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.

A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily

DyP-type peroxidases comprise a novel superfamily of heme-containing peroxidases which is unrelated to the superfamilies of known peroxidases and of which only a few members have been characterized in some detail. Here, we report the identification and characterization of a DyP-type peroxidase (TfuDyP) from the thermophilic actinomycete Thermobifida fusca. Biochemical characterization of the recombinant enzyme showed that it is a monomeric, heme-containing, thermostable, and Tat-dependently exported peroxidase. TfuDyP is not only active as dye-decolorizing peroxidase as it also accepts phenolic compounds and aromatic sulfides. In fact, it is able to catalyze enantioselective sulfoxidations, a type of reaction that has not been reported before for DyP-type peroxidases. Site-directed mutagenesis was used to determine the role of two conserved residues. D242 is crucial for catalysis while H338 represents the proximal heme ligand and is essential for heme incorporation. A genome database analysis revealed that DyP-type peroxidases are frequently found in bacterial genomes while they are extremely rare in other organisms. Most of the bacterial homologs are potential cytosolic enzymes, suggesting metabolic roles different from dye degradation. In conclusion, the detailed biochemical characterization reported here contributes significantly to our understanding of these enzymes and further emphasizes their biotechnological potential.

Kinetic mechanism of phenylacetone monooxygenase from Thermobifida fusca

Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.

Efficient Biooxidations Catalyzed by a New Generation of Self-Sufficient Baeyer―Villiger Monooxygenases

Over the last decades industrial interest in oxidative biocatalysis has increased significantly. One of the most prominent enzyme families that catalyze a variety of different oxidations are Baeyer–Villiger monooxygenases (BVMOs). These biocatalysts are part of an exclusive family of flavin-dependent enzymes that catalyze the biotransformation of aldehydes and (a)cyclic ketones to their corresponding esters and lactones. [1] Additionally, these enzymes are also known for their capability to oxidize heteroatoms (sulphur, nitrogen, boron) and perform epoxidation reactions. [2] Type I BVMOs utilize flavin adenine dinucleotide (FAD) as cofactor and NADPH as electron donor in order to activate molecular oxygen (O2) and generate a reactive C4a-peroxyflavin intermediate. This enzyme intermediate acts similarly to an organic peracid and reacts with the organic substrate and this results in formation of the oxygenated product. [3] BVMOs have been shown to carry out these oxidative reactions in a highly regio-, stereo- and enantioselective manner; this indicates that these enzymes are interesting candidates for various biocatalytic applications. [4] An obstacle of using BVMOs in a cost efficient way is the requirement of stoichiometric amounts of expensive NADPH coenzyme. Several coenzyme regeneration methods have been explored in the recent years. [5] The most efficient approach is based on the regeneration of NADPH by using a two-enzyme system (either as isolated enzyme or in whole cells). [6]

Synthesis of Chiral 3-Alkyl-3,4-dihydroisocoumarins by Dynamic Kinetic Resolutions Catalyzed by a Baeyer-Villiger Monooxygenase

Baeyer-Villiger monooxygenases have been tested in the oxidation of racemic benzofused ketones. When employing a single mutant of phenylacetone monooxygenase (M446G PAMO) under the proper reaction conditions, it was possible to achieve 3-substituted 3,4-dihydroisocoumarins with high yields and optical purities through regioselective dynamic kinetic resolution processes.

Joint-Functions of Protein Residues and Nadp(H) in Oxygen-Activation by Flavin-Containing Monooxygenase

The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP+ analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this “double-engineering” approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP+ in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.

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.

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.