Cyclohexanone monooxygenase (
EC1.14.13.22, cyclohexanone 1,2-monooxygenase, cyclohexanone oxygenase, cyclohexanone:NADPH:oxygen oxidoreductase (6-hydroxylating, 1,2-lactonizing)) is an
enzyme with
systematic namecyclohexanone,NADPH:oxygen oxidoreductase (lactone-forming).[1][2][3][4][5][6] This
enzymecatalyses the following
chemical reaction
This enzyme contains 540 residues organized into a single subunit. Cyclohexanone monooxygenase is one of the most prominent
Baeyer-Villiger monooxygenases (BVMOs) and has a low substrate specificity, allowing it to
catalyze a number of reactions; given the variety of substrates, cyclohexanone monooxygenase is a useful
enzyme for industrial applications.
Enzyme mechanism
Cyclohexanone monooxygenase (CHMO) uses
NADPH and O2 as cosubstrates and
FAD as a cofactor to insert an oxygen atom into the substrate. The process involves the formation of a falvin-peroxide and
Criegee intermediate.[7]
CHMO can also oxygenate cyclic ketones, aromatic aldehydes, and heteroatom-containing compounds.[9]
Enzyme structure
Using CHMO isolated from Rhodococcus sp. Strain HI-31 and complexed with
FAD and
NADP+, two crystal structures were obtained showing CHMO in the open and closed conformations.[7] Structurally, CHMO is stable and contains 540 residues organized into a single subunit.
CHMO contains binding domains for
NADP+ and
FAD, which are connected by two unstructured loops. The
NADP binding domain consists of the segments 152-208 and 335-380 with a helical domain constructed between residues 224-332. The helical domain shifts between the two
dinucleotide (
NADP+ and
FAD) binding domains and helps form the substrate
binding pocket. The
FAD binding domain consists of the first 140 N-terminal residues as well as residues 387-540 from the C-terminus.[7]
FAD bound to CHMO in the closed conformation (PDB ID: 3GWD).NADP+ bound to CHMO in the closed conformation (PDB ID: 3GWD). The NADP+ binding domain (residues 152-208 and 335 - 380) is shown in pink and the helical domain (residues 224-332) in yellow.
The substrate
binding pocket is well defined in the closed conformation and consists of the residues 145−146, 248, 279, 329, 434−435, 437, 492, and 507;
FAD and
NADP+ also contribute to the shape of the
binding pocket.[7]
The key distinction between the open form, CHMOopen, and the closed form, CHMOclosed, lies in the conformation of residues 487-504, which form a loop. In the closed confirmation, the loop folds upon itself, internalizing the center portion of the loop. However, in the open conformation, the loop is not visible. It is predicted that this results from the loop adopting a solvent-exposed conformation.[7]
Comparison of the open and closed CHMO structures. The closed conformation of CHMO (PDB ID: 3GWD) is shown in teal and the open conformation of CHMO (PDB ID: 3GWF) is shown in pink. CHMOclosed clearly demonstrates the loop from residues 487-504 (marked in yellow), which is not visible in CHMOopen.
Biological function
CHMO is a bacterial
flavoenzyme whose main function in the cell is to catalyze the conversion of
cyclohexanone, a cyclic
ketone, into ε-
caprolactone, a key step in the pathway for the biodedgredation of
cyclohexanol.[10] However, given the lack of specificity for CHMO, it can be used generally to form lactones from a number of four to six-membered cyclic
ketones, which can then be hydrolyzed into
aliphatic acids.[10] Moreover, CHMO has the ability to oxygenate
aromatic aldehydes and heteroatom-containing compounds – such as trivalent phosphorus and
boronic acids– as well, making it a candidate for industrial use.[10]
Industrial relevance
Utilizing its affinity for multiple substrates and given that the mechanism is one of the most well studied
Baeyer-Villiger Monooxygenases (BVMOs) with high
regio-,
chemo- and
enantioselectivity, CHMO has been identified as a useful industrial molecule.[11][7] Strain-specific primers derived from the CMHO gene have already been used to developed and optimized to both quantify and monitor levels of Lysobacter antibioticus, a potential biological disease control for crops, in agricultural soils by
PCR and real-time
qPCR.[12] With regard to the healthcare industry, CHMO mutants are a candidate for the efficient extraceullular enzymatic synthesis of (S)-
omeprazole– a drug for
gastroesophageal refux– when expressed by Pichia pastoris, a methylotrophic yeast.[13] Additionally, CHMO has demonstrated its ability to form chiral synthons making CHMO a potential target for more cost-effective drug synthesis, specifically with regard to
enantioselectivelactones.[10]
^Sheng D, Ballou DP, Massey V (September 2001). "Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis". Biochemistry. 40 (37): 11156–67.
doi:
10.1021/bi011153h.
PMID11551214.
^Stewart, J.D. (1998). "Cyclohexanone monooxygenase: a useful reagent for asymmetric Baeyer-Villiger reactions". Curr. Org. Chem. 2 (3): 195–216.
doi:
10.2174/1385272802666220128191443.
^Kayser M, Mihovilovic M, Mrstik M, Martinez C, Stewart J (1999). "Asymmetric oxidations at sulfur catalyzed by engineered strains that overexpress cyclohexanone monooxygenase". New Journal of Chemistry. 23 (8): 827–832.
doi:
10.1039/a902283j.
^Ottolina G, Bianchi S, Belloni B, Carrea G, Danieli B (1999). "First asymmetric oxidation of tertiary amines by cyclohexanone monooxygenase". Tetrahedron Lett. 40 (48): 8483–8486.
doi:
10.1016/s0040-4039(99)01780-3.
^Colonna S, Gaggero N, Carrea G, Ottolina G, Pasta P, Zambianchi F (2002). "First asymmetric epoxidation catalysed by cyclohexanone monooxygenase". Tetrahedron Lett. 43 (10): 1797–1799.
doi:
10.1016/s0040-4039(02)00029-1.