Cyclohexanone monooxygenase

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Cyclohexanone monooxygenase
Identifiers
EC no. 1.14.13.22
CAS no. 52037-90-8
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Cyclohexanone monooxygenase (EC 1.14.13.22, cyclohexanone 1,2-monooxygenase, cyclohexanone oxygenase, cyclohexanone:NADPH:oxygen oxidoreductase (6-hydroxylating, 1,2-lactonizing)) is an enzyme with systematic name cyclohexanone,NADPH:oxygen oxidoreductase (lactone-forming). [1] [2] [3] [4] [5] [6] This enzyme catalyses the following chemical reaction

Contents

cyclohexanone + NADPH + H+ + O2 hexano-6-lactone + NADP+ + H2O

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 is a member of the Baeyer-Villiger monooxygenase (BVMO) family and flavin-containing monooxygenases (FMO) superfamily. [7]

Cyclohexanone undergoes the following process, similar to the Baeyer-Villiger reactions,  to be converted into hexano-6-lactone using CHMO. [8]

  1. NADPH attaches to the active site of CHMO and transfers a hydride resulting in FADH- and NADP+.
  2. A one-electron transfer from FADH- to O2 results in a superoxide radical and FAD semiquinone.
  3. Recombination of the radical pair results in the C4a-peroxyflavin intermediate.
  4. The C4a-peroxyflavin intermediate functions as a nucleophile and attacks the cyclohexanone substrate to form the Criegee intermediate.
  5. The intermediate undergoes a rearrangement to produce the hexano-6-lactone.
  6. CHMO releases H2O and NADP+ and regenerates FADH.

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). FAD Bound to CHMO closed conformation.png
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. NADP+ Bound to CHMO closed conformation.png
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. Comparison of open and closed CHMO structures.png
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 enantioselective lactones. [10]

Related Research Articles

<span class="mw-page-title-main">Active site</span> Active region of an enzyme

In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the binding site, and residues that catalyse a reaction of that substrate, the catalytic site. Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.

<span class="mw-page-title-main">Flavin adenine dinucleotide</span> Redox-active coenzyme

In biochemistry, flavin adenine dinucleotide (FAD) is a redox-active coenzyme associated with various proteins, which is involved with several enzymatic reactions in metabolism. A flavoprotein is a protein that contains a flavin group, which may be in the form of FAD or flavin mononucleotide (FMN). Many flavoproteins are known: components of the succinate dehydrogenase complex, α-ketoglutarate dehydrogenase, and a component of the pyruvate dehydrogenase complex.

<span class="mw-page-title-main">Baeyer–Villiger oxidation</span> Organic reaction

The Baeyer–Villiger oxidation is an organic reaction that forms an ester from a ketone or a lactone from a cyclic ketone, using peroxyacids or peroxides as the oxidant. The reaction is named after Adolf von Baeyer and Victor Villiger who first reported the reaction in 1899.

<span class="mw-page-title-main">Aldose reductase</span> Enzyme

In enzymology, aldose reductase is a cytosolic NADPH-dependent oxidoreductase that catalyzes the reduction of a variety of aldehydes and carbonyls, including monosaccharides. It is primarily known for catalyzing the reduction of glucose to sorbitol, the first step in polyol pathway of glucose metabolism.

In enzymology, a 4-hydroxyacetophenone monooxygenase (EC 1.14.13.84) is an enzyme that catalyzes the chemical reaction:

<span class="mw-page-title-main">4-hydroxybenzoate 3-monooxygenase</span> Flavoprotein

The enzyme 4-hydroxybenzoate 3-monooxygenase, also commonly referred to as para-hydroxybenzoate hydroxylase (PHBH), is a flavoprotein belonging to the family of oxidoreductases. Specifically, it is a hydroxylase, and is one of the most studied enzymes and catalyzes reactions involved in soil detoxification, metabolism, and other biosynthetic processes.

<span class="mw-page-title-main">4-Hydroxyphenylacetate 3-monooxygenase</span> Class of enzymes

4-hydroxyphenylacetate 3-monooxygenase (EC 1.14.14.9) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Cholesterol 24-hydroxylase</span> Protein family

Cholesterol 24-hydroxylase, also commonly known as cholesterol 24S-hydroxylase, cholesterol 24-monooxygenase, CYP46, or CYP46A1, is an enzyme that catalyzes the conversion of cholesterol to 24S-hydroxycholesterol. It is responsible for the majority of cholesterol turnover in the human central nervous system. The systematic name of this enzyme class is cholesterol,NADPH:oxygen oxidoreductase (24-hydroxylating).

<span class="mw-page-title-main">Kynurenine 3-monooxygenase</span> Enzyme

In enzymology, a kynurenine 3-monooxygenase (EC 1.14.13.9) is an enzyme that catalyzes the chemical reaction

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In enzymology, a ferredoxin-NADP+ reductase (EC 1.18.1.2) abbreviated FNR, is an enzyme that catalyzes the chemical reaction

Flavin reductase a class of enzymes. There are a variety of flavin reductases, which bind free flavins and through hydrogen bonding, catalyze the reduction of these molecules to a reduced flavin. Riboflavin, or vitamin B, and flavin mononucleotide are two of the most well known flavins in the body and are used in a variety of processes which include metabolism of fat and ketones and the reduction of methemoglobin in erythrocytes. Flavin reductases are similar and often confused for ferric reductases because of their similar catalytic mechanism and structures.

Ketosteroid monooxygenase (EC 1.14.13.54, steroid-ketone monooxygenase, progesterone, NADPH2:oxygen oxidoreductase (20-hydroxylating, ester-producing), 17alpha-hydroxyprogesterone, NADPH2:oxygen oxidoreductase (20-hydroxylating, side-chain cleaving), androstenedione, NADPH2:oxygen oxidoreductase (17-hydroxylating, lactonizing)) is an enzyme with systematic name ketosteroid,NADPH:oxygen oxidoreductase (20-hydroxylating, ester-producing/20-hydroxylating, side-chain cleaving/17-hydroxylating, lactonizing). This enzyme catalyses the following chemical reaction

Monocyclic monoterpene ketone monooxygenase (EC 1.14.13.105, 1-hydroxy-2-oxolimonene 1,2-monooxygenase, dihydrocarvone 1,2-monooxygenase, MMKMO) is an enzyme with systematic name (-)-menthone,NADPH:oxygen oxidoreductase. This enzyme catalyses the following chemical reaction

(2,2,3-Trimethyl-5-oxocyclopent-3-enyl)acetyl-CoA 1,5-monooxygenase (EC 1.14.13.160, 2-oxo-Delta3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase, 2-oxo-Delta3-4,5,5-trimethylcyclopentenylacetyl-CoA 1,2-monooxygenase, OTEMO) is an enzyme with systematic name ((1R)-2,2,3-trimethyl-5-oxocyclopent-3-enyl)acetyl-CoA,NADPH:oxygen oxidoreductase (1,5-lactonizing). This enzyme catalyses the following chemical reaction

Neopentalenolactone D synthase (EC 1.14.13.171, ptlE (gene)) is an enzyme with systematic name 1-deoxy-11-oxopentalenate,NADH:oxygen oxidoreductase (neopentalenolactone-D forming). This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">Flavin-containing monooxygenase</span> Class of enzymes

The flavin-containing monooxygenase (FMO) protein family specializes in the oxidation of xeno-substrates in order to facilitate the excretion of these compounds from living organisms. These enzymes can oxidize a wide array of heteroatoms, particularly soft nucleophiles, such as amines, sulfides, and phosphites. This reaction requires an oxygen, an NADPH cofactor, and an FAD prosthetic group. FMOs share several structural features, such as a NADPH binding domain, FAD binding domain, and a conserved arginine residue present in the active site. Recently, FMO enzymes have received a great deal of attention from the pharmaceutical industry both as a drug target for various diseases and as a means to metabolize pro-drug compounds into active pharmaceuticals. These monooxygenases are often misclassified because they share activity profiles similar to those of cytochrome P450 (CYP450), which is the major contributor to oxidative xenobiotic metabolism. However, a key difference between the two enzymes lies in how they proceed to oxidize their respective substrates; CYP enzymes make use of an oxygenated heme prosthetic group, while the FMO family utilizes FAD to oxidize its substrates.

<span class="mw-page-title-main">Mercury(II) reductase</span>

Mercury(II) reductase (EC 1.16.1.1), commonly known as MerA, is an oxidoreductase enzyme and flavoprotein that catalyzes the reduction of Hg2+ to Hg0. Mercury(II) reductase is found in the cytoplasm of many eubacteria in both aerobic and anaerobic environments and serves to convert toxic mercury ions into relatively inert elemental mercury.

<span class="mw-page-title-main">L-ornithine N5 monooxygenase</span> Enzyme

L-ornithine N5 monooxygenase (EC 1.14.13.195 or EC 1.14.13.196) is an enzyme which catalyzes one of the following chemical reactions:

L-ornithine + NADPH + O2 N(5)-hydroxy-L-ornithine + NADP+ + H2O L-ornithine + NAD(P)H + O2 N(5)-hydroxy-L-ornithine + NAD(P)+ + H2O

<span class="mw-page-title-main">Marco Fraaije</span> Dutch scientist

Marco Wilhelmus Fraaije is a Dutch scientist whose research concerns enzymology of redox enzymes, enzyme discovery & engineering and biocatalysis at the Groningen Biomolecular Sciences and Biotechnology Institute (GBB) at the University of Groningen.

References

  1. ↑ Donoghue NA, Norris DB, Trudgill PW (March 1976). "The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871". European Journal of Biochemistry. 63 (1): 175–92. doi: 10.1111/j.1432-1033.1976.tb10220.x . PMID   1261545.
  2. ↑ 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. PMID   11551214.
  3. ↑ 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.
  4. ↑ 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.
  5. ↑ 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.
  6. ↑ 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.
  7. 1 2 3 4 5 6 7 Mirza, I. Ahmad; Yachnin, Brahm J.; Wang, Shaozhao; Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Iwaki, Hiroaki; Hasegawa, Yoshie; Lau, Peter C. K.; Berghuis, Albert M. (2009-07-01). "Crystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding Cofactor". Journal of the American Chemical Society. 131 (25): 8848–8854. doi:10.1021/ja9010578. ISSN   0002-7863. PMID   19385644. S2CID   207138937.
  8. ↑ Fordwour, Osei Boakye; Wolthers, Kirsten R. (2018-12-01). "Active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase". Archives of Biochemistry and Biophysics. 659: 47–56. doi:10.1016/j.abb.2018.09.025. ISSN   0003-9861. PMID   30287236. S2CID   52919877.
  9. ↑ Sheng, D.; Ballou, D. P.; Massey, V. (2001-09-18). "Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis". Biochemistry. 40 (37): 11156–11167. doi:10.1021/bi011153h. ISSN   0006-2960. PMID   11551214.
  10. 1 2 3 4 Sheng, Dawei; Ballou, David P.; Massey, Vincent (2001-09-01). "Mechanistic Studies of Cyclohexanone Monooxygenase: Chemical Properties of Intermediates Involved in Catalysis". Biochemistry. 40 (37): 11156–11167. doi:10.1021/bi011153h. ISSN   0006-2960. PMID   11551214.
  11. ↑ Beek, Hugo L. van; Gonzalo, Gonzalo de; Fraaije, Marco W. (2012-03-05). "Blending Baeyer–Villiger monooxygenases: using a robust BVMO as a scaffold for creating chimeric enzymes with novel catalytic properties". Chemical Communications. 48 (27): 3288–3290. doi:10.1039/C2CC17656D. ISSN   1364-548X. PMID   22286124.
  12. ↑ Lina, Fu; Ting, Wang; Lanfang, Wei; Jun, Yang; Qi, Liu; Yating, Wang; Xing, Wang; Guanghai, Ji (2018). "Specific detection of Lysobacter antibioticus strains in agricultural soil using PCR and real-time PCR". FEMS Microbiology Letters. 365 (20). doi: 10.1093/femsle/fny219 . PMID   30202922.
  13. ↑ Li, Ya-Jing; Zheng, Yu-Cong; Geng, Qiang; Liu, Feng; Zhang, Zhi-Jun; Xu, Jian-He; Yu, Hui-Lei (2021-08-27). "Secretory expression of cyclohexanone monooxygenase by methylotrophic yeast for efficient omeprazole sulfide bio-oxidation". Bioresources and Bioprocessing. 8 (1): 81. doi: 10.1186/s40643-021-00430-1 . ISSN   2197-4365. S2CID   237330516.
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