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

Microbiological transformation 35: Enantioselective one-step preparative scale synthesis of 1,3-dithiane-1-oxide via whole-cell bacterial oxidation

This work describes the preparative scale enantioselective oxidation of 1,3-dithiane to the corresponding monosulfoxide using whole-cell cultures of two bacteria, i.e. Acinetobacter calcoaceticus NCIMB 9871 and Pseudomonas sp. NCIMB 9872

Microbiological transformations 36: Preparative scale synthesis of chiral thioacetal and thioketal sulfoxides using whole-cell biotransformations

This work describes the preparative scale enantioselective oxidation of some prochiral dithioacetals and dithioketals to their corresponding chiral monosulfoxides using whole-cell cultures of microorganisms

Broadening the scope of Baeyer–Villiger monooxygenase activities toward α,β-unsaturated ketones: a promising route to chiral enol-lactones and ene-lactones

International audienc

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one‐pot/two‐step biocatalytic whole‐cell system

International audienceThe efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce -caprolactone and 3,4dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of E. coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counterbalanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain a complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g -caprolactone, for a productivity of 244 mg. L-1 .h-1) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for a productivity of 140 mg. L-1 .h-1)