Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

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

The Substrate-Bound Crystal Structure of a Baeyer–Villiger Monooxygenase Exhibits a Criegee-like Conformation

The Baeyer\u2013Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer\u2013Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP+ and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP+ and FAD, to 2.4 \uc5 resolution. This structure shows a drastic rotation of the NADP+ cofactor in comparison to previously reported NADP+-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP+ permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme\u2019s chemospecificity, to a location above the flavin\u2013peroxide adduct where catalysis occurs.Peer reviewed: YesNRC publication: Ye

Contribution à la conception des architectures réseaux (application aux systèmes embarqués sur une seule puce)

La technologie de fabrication des circuits intégrés a permis de passer des composants spécifiques ASIC (Application Specific Integrated Circuits) aux systèmes embarqués sur une seule puce SoC (System-on-Chip). Grâce à cette évolution, les SoC peuvent intégrer sur la même puce plusieurs processeurs, de la mémoire, différents blocs IP (Intellectual Property), ainsi que des réseaux de communication complexes. Cette volonté d'intégration et de miniaturisation des systèmes intégrés requiert une adéquation entre l'application et le réseau de communication. Cette adéquation opère sur deux axes complémentaires qui sont : le développement de réseau de communication qui tient compte des besoins des IP en termes de QoS et l'adaptation au niveau des interfaces. Dans ce contexte, nous nous sommes proposé de mettre en œuvre l'adéquation du couple application/réseau. L'étude d'un exemple nous a ainsi permis d'améliorer la qualité visuelle des séquences vidéo de type MPEG2 à travers le réseau à haut débit ATM. Nous avons également contribué à la généralisation cette approche d'adéquation aux systèmes embarqués sur une seule puce. Cette généralisation à été mise en œuvre par le biais du développement d'une architecture d'un système de communication intégré basé sur l'utilisation de la technique de routage de type "wormhole" pour le NoC intégré ainsi que la conception de deux adaptateurs : AHB/NoC/AHB et Avalon/NoC/RAM. Cette étude a été validée à travers le prototypage de l'architecture du NoC proposée sur une plateforme du type Stratix II d'AlteraThe manufacturing technology of the integrated circuits made it possible to pass from specific components ASIC (Application Specific Integrated Circuits) to the embedded systems on chip SoC (System-on-Chip). Due to this evolution, SoCs can contain several processors, memories, various blocks of IP (Intellectual Property), and complex networks of communication. In order to achieve a high degree of integration and miniaturization, we have to make an adequation between the application and communication network taking into account the QoS needs for the IP through the communication network. In this context, we studied experiment adequation of the couple application/network through two examples. The first one aims to improve visual quality of the MPEG-2 video sequences through the ATM network. The second one was a contribution to the generalisation of the adequation approach for the embedded systems on chip. The corresponding architecture including two adapters (AHB/NoC/AHB and the Avalon/NoC/RAM) has been implemented on the Platform StratixII of AlteraLORIENT-BU (561212106) / SudocSudocFranceF