Hybrid catalysis for enantioselective Baeyer-Villiger oxidation and stereoselective epoxidation: a Cp*Ir complex to fuel FMN and FAD reduction for flavoprotein monooxygenase modules

Taking advantage of the unique properties of two-component flavo-monooxygenases and the ability of [Cp*Ir(bpy-OMe)H]+ to transfer hydrides to reduce flavins, we extended the scope of the pH- and oxygen-robust iridium(iii)-complex to drive the enzymatic reaction of a FMN-dependent Baeyer-Villiger monooxygenase and a FAD-dependent styrene monooxygenase (respectively FPMO Group C and E), using formic acid as H-donor for NADH recycling

Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMOXeno) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMOAcineto) in Synechocystis sp. PCC 6803 (25 vs 2.3 U g(DCW)(-1) at an optical density of OD750 = 10) and an initial rate of 3.7 +/- 0.2 mM h(-1). While the cells containing CHMOAcineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMOXeno, which was attributed to the much faster lactone formation and a 10-fold lower KM value of BVMOXeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h-1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygendependent enzymatic processes

Evolution study of the Baeyer-Villiger monooxygenases enzyme family: functional importance of the highly conserved residues.

International audienceBaeyer-Villiger monooxygenases (BVMOs) catalyze the transformation of linear and cyclic ketones into their corresponding esters and lactones by introducing an oxygen atom into a C-C bond. This bioreaction has numerous advantages compared to its chemical version; it does not induce the use of potentially harmful reagents (i.e., green chemistry) and displays significant better enantio- and regio-selectivity. New potential BVMOs were searched using sequence homology for type I BVMO proteins. 116 new sequences were identified as new putative BVMOs respecting the defined selection criteria. Multiple sequence alignments were carried out on the selected sequences to study the conservation of structurally and/or functionally important amino acids during evolution. Type I BVMO signature motif was found to be conserved in 94.8% of the sequences. We noticed also the highly conserved - but previously unnoticed - Threonine 167 (93.1%), located in the signature motif; this position could be added in the pattern used to characterize specific Type I enzymes. Amino acids at the vicinity of the FAD and NADPH cofactors were found also to be highly conserved and the details of the interactions were emphasized. Interestingly, residues at the enzyme binding site were found less conserved in terms of sequence evolution, leading sometimes to some important amino acid changes. These behaviors could explain the enzyme selectivity and specificity for different ligands

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer–Villiger oxidations

International audienceTwo-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer-Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH 2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH 2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer-Villiger oxidation of racbicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case. † Electronic supplementary information (ESI) available. Se

Auto-sufficient enzymatic cascade combining a non selective and promiscuous ADH and BVMOS: Application for dihydrocoumarin synthesis

International audienceDihydrocoumarin is a plant metabolite widely used as flavoring agent in agro-food industries and also as common fragrance in cosmetics. As a lot of flavor chemicals, dihydrocoumarin is found in minor quantities in its natural plant sources, the sweet clover (Melilotus officinalis) and tonka beans (Dipteryx odorata) so processes are needed to produce this natural flavor in a efficient and eco-friendly manner. Baeyer-Villiger MonoOxygenases (BVMOs) are well known flavoenzymes able to transform efficiently ketone into ester or lactone with high regio-and stereoselectivities. Nevertheless, BVMOs are strictly NADPH-dependent, and therefore require a stoichiometric amount of the expensive nicotinamide cofactor. To address this issue, the multi-enzyme syntheses provide the opportunity to generate efficient auto-sufficient systems and only a limited number of such systems involving BVMOs has been reported to date. We will present here a new efficient access to dihydrocoumarin via a two-enzyme mediated oxidation of Indanol involving Alcohol Dehydrogenase (ADH) and BVMO. The originality of our approach comes from the features of ADH, an enzyme discovered from the biodiversity via a dedicated High Throughput Screening. The ADH is a NADP-dependent and non-enantioselective enzyme which enables on the one hand cofactor recycling and on the other hand a complete transformation of the racemic alcohol. Moreover, it presents a quite large scope of substrates. Several plasmid constructions and combinations have been tested first for the formation of e-caprolactone and compared in order to elaborate a versatile platform using ADH and different BVMOs. Then to optimize the formation of Dihydrocoumarin and limit the hydrolysis, we investigated the composition of the biotransformation medium in whole-cell process as well as in purified enzyme system.Still exploring the diversity of BVMOs activities, we wish to expand the range of potentially valued compounds obtained using these systems. (Ménil et al.

Auto-sufficient enzymatic cascade combining a non selective and promiscuous ADH and BVMOS: Application for dihydrocoumarin synthesis

Dihydrocoumarin is a plant metabolite widely used as flavoring agent in agro-food industries and also as common fragrance in cosmetics. As a lot of flavor chemicals, dihydrocoumarin is found in minor quantities in its natural plant sources, the sweet clover (Melilotus officinalis) and tonka beans (Dipteryx odorata) so processes are needed to produce this natural flavor in a efficient and eco-friendly manner. Baeyer-Villiger MonoOxygenases (BVMOs) are well known flavoenzymes able to transform efficiently ketone into ester or lactone with high regio-and stereoselectivities. Nevertheless, BVMOs are strictly NADPH-dependent, and therefore require a stoichiometric amount of the expensive nicotinamide cofactor. To address this issue, the multi-enzyme syntheses provide the opportunity to generate efficient auto-sufficient systems and only a limited number of such systems involving BVMOs has been reported to date. We will present here a new efficient access to dihydrocoumarin via a two-enzyme mediated oxidation of Indanol involving Alcohol Dehydrogenase (ADH) and BVMO. The originality of our approach comes from the features of ADH, an enzyme discovered from the biodiversity via a dedicated High Throughput Screening. The ADH is a NADP-dependent and non-enantioselective enzyme which enables on the one hand cofactor recycling and on the other hand a complete transformation of the racemic alcohol. Moreover, it presents a quite large scope of substrates. Several plasmid constructions and combinations have been tested first for the formation of e-caprolactone and compared in order to elaborate a versatile platform using ADH and different BVMOs. Then to optimize the formation of Dihydrocoumarin and limit the hydrolysis, we investigated the composition of the biotransformation medium in whole-cell process as well as in purified enzyme system.Still exploring the diversity of BVMOs activities, we wish to expand the range of potentially valued compounds obtained using these systems. (Ménil et al.

Auto-sufficient enzymatic cascade combining a non selective and promiscuous ADH and BVMOS: Application for dihydrocoumarin synthesis

Dihydrocoumarin is a plant metabolite widely used as flavoring agent in agro-food industries and also as common fragrance in cosmetics. As a lot of flavor chemicals, dihydrocoumarin is found in minor quantities in its natural plant sources, the sweet clover (Melilotus officinalis) and tonka beans (Dipteryx odorata) so processes are needed to produce this natural flavor in a efficient and eco-friendly manner. Baeyer-Villiger MonoOxygenases (BVMOs) are well known flavoenzymes able to transform efficiently ketone into ester or lactone with high regio-and stereoselectivities. Nevertheless, BVMOs are strictly NADPH-dependent, and therefore require a stoichiometric amount of the expensive nicotinamide cofactor. To address this issue, the multi-enzyme syntheses provide the opportunity to generate efficient auto-sufficient systems and only a limited number of such systems involving BVMOs has been reported to date. We will present here a new efficient access to dihydrocoumarin via a two-enzyme mediated oxidation of Indanol involving Alcohol Dehydrogenase (ADH) and BVMO. The originality of our approach comes from the features of ADH, an enzyme discovered from the biodiversity via a dedicated High Throughput Screening. The ADH is a NADP-dependent and non-enantioselective enzyme which enables on the one hand cofactor recycling and on the other hand a complete transformation of the racemic alcohol. Moreover, it presents a quite large scope of substrates. Several plasmid constructions and combinations have been tested first for the formation of e-caprolactone and compared in order to elaborate a versatile platform using ADH and different BVMOs. Then to optimize the formation of Dihydrocoumarin and limit the hydrolysis, we investigated the composition of the biotransformation medium in whole-cell process as well as in purified enzyme system.Still exploring the diversity of BVMOs activities, we wish to expand the range of potentially valued compounds obtained using these systems. (Ménil et al.

Silica-Catalysed and Highly Stereoselective Convergent and Nonconvergent Rearrangements of Menthone Enol Acetate Epoxides: Easy Access to the Four α-Hydroxymenthone Stereoisomers

International audienc