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

Abstract

The Baeyer–Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer–Villiger 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 Å 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’s chemospecificity, to a location above the flavin–peroxide adduct where catalysis occurs.

Lactone-Bound Structures of Cyclohexanone Monooxygenase Provide Insight into the Stereochemistry of Catalysis

“…It has been previously noted that the CHMO Rotated structure, which is in an ideal position for formation of the Criegee intermediate, does not place cyclohexanone close to these residues. 28 In contrast, both CHMO Tight and CHMO Loose place ε-caprolactone in close proximity to these key residues (Figure 3). Assuming that the substrate, in the presence of the peroxyanion intermediate, would also bind in the same manner as the product in our structures, this strongly suggests that the acceptance of substrates, as well as the regio-and enantiospecificity of the enzyme, is determined in the Loose and/or Tight conformations.…”

“…Combining the new CHMO Tight and CHMO Loose crystal structures with the previously reported CHMO Rotated structure, we now have a crystal structure with either substrate or product bound for each of the six critical substrate-and product-bound states proposed in the BVMO structural mechanism. 28 These are also all structures of the same enzyme, allowing them to be compared directly. The CHMO Rotated crystal structure corresponds to the Criegee-like states (states F and G), which are characterized by a closed off binding pocket that is large enough to accommodate a wide range of larger substrates.…”

Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism

“…The crystal structure of CHMO was reported several years ago and this highlighted multiple conformations of the bound nicotinamide coenzyme required to reduce the flavin and stabilize formation of the Criegee intermediate formed in the reaction cycle [30]. More recent studies have reported the structure of a CHMO complex with NADP + and 2-cyclohexenone and reveal major rotation of the NADP + cofactor away from the flavin cofactor so that it is no longer stacked with the tricyclic isoalloxazine ring [31]. In this new conformation, cyclohexanone is now located over the flavin in a position that enables formation of the Criegee intermediate.…”

Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation

“…Previous structural as well as mutagenesis studies have concluded that the C4a-(hydro)peroxyflavin intermediate is stabilized by NADP + which remains bound until the last step of the catalytic cycle [8,[29][30][31][32]. However, if this intermediate is not stabilized or the NADP + binding is compromised, the uncoupling of the enzyme results in hydrogen peroxide release.…”