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Literature summary extracted from

  • Mirza, I.A.; Yachnin, B.J.; Wang, S.; Grosse, S.; Bergeron, H.; Imura, A.; Iwaki, H.; Hasegawa, Y.; Lau, P.C.; Berghuis, A.M.
    Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor (2009), J. Am. Chem. Soc., 131, 8848-8854.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.22 gene amplification, cloning in pUC19 vector, transformation in Escherichia coli DH5 of plasmid pCMR100 containing the complete gene chnB1 (1620 bp), amplification of fragment carrying chnB1, cloning into plasmid pSD80 to result in pSDRmchnB1, overexpressed in Escherichia coli BL21 (DE3) Rhodococcus sp. HI-31

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.13.22 enzyme in complex with FAD and NADP+ in 2 distinct states, hanging drop or sitting drop vapor diffusion methods, crystal form I with 0.2 M sodium acetate trihydrate, 0.1 M sodium cacodylate pH 6.5, and 30% PEG 8000, for crystal form II with 0.2 M magnesium acetate tetrahydrate, 0.1 M sodium cacodylate pH 6.5, and 20% PEG 8000 Rhodococcus sp. HI-31

Protein Variants

EC Number Protein Variants Comment Organism
1.14.13.22 W492A 14% activity with cyclohexanone compared to wild type enzyme Rhodococcus sp. HI-31

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.13.22 NADP+
-
 Rhodococcus sp. HI-31

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.22 0.006
-
 NADPH mutant W492A, 0.1 mM cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 25°C Rhodococcus sp. HI-31
1.14.13.22 0.014
-
 NADPH wild type enzyme, 0.1 mM cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 25°C Rhodococcus sp. HI-31

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.22 cyclohexanone + NADPH + H+ + O2 Rhodococcus sp. HI-31
-
 hexano-6-lactone + NADP+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.22 Rhodococcus sp. HI-31 C0STX7
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.22 crude extract of Rhodococcus cells is partially purified using ammonium sulfate fractionation (50-70%), chromatography on Butyl-Toyopearl 650S with a linear gradient of 30%-0% (NH4)2SO4, and gel filtration on a Sephadex G-150 column. Purification of the recombinant enzyme: cells centrifuged and lysed with a French press, crude extract centrifuged, supernatant loaded on a DEAE-Sepharose FF column equilibrated with 50 mM sodium phosphate buffer (pH 7.0), elution with 0-0.2 M NaCl gradient, ammonium sulfate added to active fractions (30%), loaded onto a Butyl-S-Sepharose 6 FF column equilibrated with 30% ammonium sulfate in 50 mM sodium phosphate buffer (pH 7.0), elution with 30-0% ammonium sulfate gradient, active fractions pooled, concentrated, applied to two combined Superose 6 HR (10/300)/Superose 12 HR (10/300) gel filtration columns equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl, elution with same buffer, pooled, concentrated with YM 10 membrane Rhodococcus sp. HI-31

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.13.22 0.85
-
 mutant W492A, cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 0.1 mM NADPH, 25°C Rhodococcus sp. HI-31
1.14.13.22 6
-
 wild type enzyme, cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 0.1 mM NADPH, 25°C Rhodococcus sp. HI-31

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.22 (2R,6S)-2,6-dimethylcyclohexanone + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 cis-3, 7-dimethyl-2-oxepanone + NADP+ + H2O
-
 ?
1.14.13.22 1,4-dioxaspiro[4.5]decan-8-one + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 1,4,8-trioxa-spiro[4,6]undecan-9-one + NADP+ + H2O
-
 ?
1.14.13.22 2,2,6-trimethylcyclohexanone + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 3,7,7-trimethyloxepan-2-one + NADP+ + H2O
-
 ?
1.14.13.22 2-hexylcyclopentanone + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 6-hexyl-tetrahydro-pyran-2-one + NADP+ + H2O
-
 ?
1.14.13.22 2-phenylcyclohexanone + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 3-phenyloxepan-2-one + NADP+ + H2O
-
 ?
1.14.13.22 3,3,5-trimethylcyclohexanone + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 4,6,6-trimethyloxepan-2-one + 4,4,6-trimethyloxepan-2-one + NADP+ + H2O
-
 ?
1.14.13.22 3,4-dihydronaphthalen-2(1H)-one + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 8,9-dihydro-5H-7-oxa-benzocyclo-hepten-6-one + NADP+ + H2O
-
 ?
1.14.13.22 4-tert-butylcyclohexanone + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 5-t-butyl-2-oxepanone + NADP+ + H2O
-
 ?
1.14.13.22 cyclohexanone + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 hexano-6-lactone + NADP+ + H2O
-
 ?
1.14.13.22 octahydronaphthalen-1(2H)-one + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 octahydro-benzo[b]oxepin-2-one + octahydro-benzo[c]oxepin-1-one + NADP+ + H2O
-
 ?
1.14.13.22 octahydronaphthalen-2(1H)-one + NADPH + H+ + O2
-
 Rhodococcus sp. HI-31 cis-octahydro-benzo[c]oxepin-3-one + trans-octahydro-benzo[c]oxepin-3-one + cis-octahydro-7-oxa-benzocyclohepten-6-one + NADP+ + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.13.22 monomer 1 * x, 540 residues, 0.94 mol FAD per mol enzyme Rhodococcus sp. HI-31

Synonyms

EC Number Synonyms Comment Organism
1.14.13.22 CHMO 2 cyclohexanone monooxygenase related genes chnB1 and chnB2 Rhodococcus sp. HI-31
1.14.13.22 chnB1 protein
-
 Rhodococcus sp. HI-31
1.14.13.22 cyclohexanone monooxygenase
-
 Rhodococcus sp. HI-31

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.22 FAD
-
 Rhodococcus sp. HI-31
1.14.13.22 NADPH
-
 Rhodococcus sp. HI-31

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.14.13.22 0.115
-
 NADP+ wild type enzyme, 0.1 mM cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 25°C Rhodococcus sp. HI-31
1.14.13.22 0.39
-
 NADP+ mutant W492A, 0.1 mM cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 25°C Rhodococcus sp. HI-31

General Information

EC Number General Information Comment Organism
1.14.13.22 metabolism flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones, size and shape of substrate binding pocket are adjustable to accommodate a diverse range of substrates Rhodococcus sp. HI-31