Cloned (Comment) | Organism |
---|---|
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 (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
W492A | 14% activity with cyclohexanone compared to wild type enzyme | Rhodococcus sp. HI-31 |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Rhodococcus sp. HI-31 |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.006 | - |
NADPH | mutant W492A, 0.1 mM cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 25°C | Rhodococcus sp. HI-31 | |
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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
cyclohexanone + NADPH + H+ + O2 | Rhodococcus sp. HI-31 | - |
hexano-6-lactone + NADP+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodococcus sp. HI-31 | C0STX7 | - |
- |
Purification (Comment) | Organism |
---|---|
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 Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.85 | - |
mutant W492A, cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 0.1 mM NADPH, 25°C | Rhodococcus sp. HI-31 |
6 | - |
wild type enzyme, cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 0.1 mM NADPH, 25°C | Rhodococcus sp. HI-31 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(2R,6S)-2,6-dimethylcyclohexanone + NADPH + H+ + O2 | - |
Rhodococcus sp. HI-31 | cis-3, 7-dimethyl-2-oxepanone + NADP+ + H2O | - |
? | |
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 | - |
? | |
2,2,6-trimethylcyclohexanone + NADPH + H+ + O2 | - |
Rhodococcus sp. HI-31 | 3,7,7-trimethyloxepan-2-one + NADP+ + H2O | - |
? | |
2-hexylcyclopentanone + NADPH + H+ + O2 | - |
Rhodococcus sp. HI-31 | 6-hexyl-tetrahydro-pyran-2-one + NADP+ + H2O | - |
? | |
2-phenylcyclohexanone + NADPH + H+ + O2 | - |
Rhodococcus sp. HI-31 | 3-phenyloxepan-2-one + NADP+ + H2O | - |
? | |
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 | - |
? | |
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 | - |
? | |
4-tert-butylcyclohexanone + NADPH + H+ + O2 | - |
Rhodococcus sp. HI-31 | 5-t-butyl-2-oxepanone + NADP+ + H2O | - |
? | |
cyclohexanone + NADPH + H+ + O2 | - |
Rhodococcus sp. HI-31 | hexano-6-lactone + NADP+ + H2O | - |
? | |
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 | - |
? | |
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 | Comment | Organism |
---|---|---|
monomer | 1 * x, 540 residues, 0.94 mol FAD per mol enzyme | Rhodococcus sp. HI-31 |
Synonyms | Comment | Organism |
---|---|---|
CHMO | 2 cyclohexanone monooxygenase related genes chnB1 and chnB2 | Rhodococcus sp. HI-31 |
chnB1 protein | - |
Rhodococcus sp. HI-31 |
cyclohexanone monooxygenase | - |
Rhodococcus sp. HI-31 |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Rhodococcus sp. HI-31 | |
NADPH | - |
Rhodococcus sp. HI-31 |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.115 | - |
NADP+ | wild type enzyme, 0.1 mM cyclohexanone, 50 mM Tris-HCl buffer (pH 9.0), 25°C | Rhodococcus sp. HI-31 | |
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 | Comment | Organism |
---|---|---|
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 |