Literature summary for 1.14.13.22 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.

Search for more literature for 1.14.13.22

Cloned(Commentary)

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 (Commentary)

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

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

Inhibitors

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

KM Value [mM]

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Rhodococcus sp. HI-31	C0STX7	-	-

Purification (Commentary)

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

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 [micromol/min/mg]

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(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

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

Synonyms

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

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

Ki Value [mM]

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

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