• Bulletin of the Korean Chemical Society
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• v.29 no.12
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• pp.2491-2495
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• 2008

• YAKHAK HOEJI
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• v.37 no.1
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• pp.95-99
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• 1993

Peroxidase activity of cytochrome P-450 was examined using N, N-dimethylaniline (NDA) as a substrate and cumene hydroperoxide (CHP) as an oxidant. The initial rates of the N-demethylation for varied concentrations of NDA (0.05-0.5 mM) by P-450 at different fixed concentrations of CHP (0.02-0.2 mM) were determined. The results suggest that P-450 proceeds its peroxidative reaction by the rapid equilibrium random bi bi mechanism to form a ternary complex with substrate and oxidant as an active intermediate.

• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.956-964
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• 2017

Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steady-state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The $k_m$ and $k_{cat}$ values for compactin were $39.1{\pm}8.8{\mu}M$ and $1.12{\pm}0.09min^{-1}$, respectively. The $k_{cat}/K_m$ value for compactin ($0.029min^{-1}{\cdot}{\mu}M^{-1}$) was lower than that for diclofenac ($0.114min^{-1}{\cdot}{\mu}M^{-1}$). Spectroscopic analysis showed that CYP105D7 binds to compactin with a $K_d$ value of $17.5{\pm}3.6{\mu}M$. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

• Toxicological Research
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• v.15 no.1
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• pp.95-101
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• 1999

Cytochrome P450 (CYP) 3A4 metabolizes aflatoxin B1 (AFB1) to AFB1-exo-8,9-epoxide (8,9-epoxidation) and aflatoxin Q1 (AFQ1; 3$\alpha$-hydroxylation) simultaneously. We investigated whether each metabolite was formed via its own binding site of CAP3A4 active site. Kinetics of the formation of the two metabolites were sigmoidal and consistent with the kinetics of substrate activation. The HIll model predicted that two substrate binding wites are involved in the oxidationof AFB1 by CYP3A4. Dehydronifedipine, a metabolite of nifedipine generated by CYP3A4, inhibited the formation of AFQ1 without any inhibition in the formation of AFB1-exo-8,9-epoxidation. Dehydronifedipine was found to act as a reversible competitive inhibitor against 3$\alpha$-hydroxylation of AFB1. Vmax and S0.5 of the 8,9-epoxidation were not changed in the presence of 0, 50, or 100 $\mu\textrm{M}$ dehydronifedipine. S0.5 of 3$\alpha$-hydroxylation was increased from 58$\pm$4 $\mu\textrm{M}$ to 111$\pm$8 $\mu\textrm{M}$ in the presence of 100 $\mu\textrm{M}$ nifedipine whereas Vmax was not changed. These results suggest that there exist two independent binding sites in the active site of CAP3A4 . One binding site is responsible for AFB1-exo-8,9-epoxidation and the other is involved in 3$\alpha$-hydroxylation of AFB1. Dehydronifedipine might selectively bind to the site which is responsible for the formation of AFQ1 in the active site of CYP3A4.

• Biomolecules & Therapeutics
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• v.21 no.6
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• pp.487-492
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• 2013

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

• Korean Chemical Engineering Research
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• v.44 no.4
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• pp.387-392
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• 2006

(Fe, Co)/zeolite catalysts such as (Fe, Co)/NaY, (Fe, Co)/NaBeta and (Fe, Co)/HUSY were prepared by ion-exchange method and their catalytic performance was examined in the hydroxylation of phenol with $H_2O_2$ for the production of catechol. The (Fe, Co)/NaBeta catalyst showed its best performance at reaction temperature=$70^{\circ}C$, molar ratio of phenol/$H_2O_2=3$, weight ratio of phenol/catalyst=50 and weight ratio of solvent (water)/phenol=6 as 20％ of phenol conversion, 77％ of the selectivity for the hydroxylation, 70％ of the selectivity for catechol, and 2.5 of the formation ratio of catechol/hydroquinone. The (Fe, Co)/zeolite catalysts showed the reproducible activities without deactivation after repeated regeneration. The fresh and used(Fe, Co)/zeolites were characterized by XRD, UV-VIS DRS, and XPS and their catalytic performance was discussed based on these characterization results.

• Microbiology and Biotechnology Letters
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• v.38 no.4
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• pp.475-480
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• 2010

Actinomycetes are Gram-positive soil bacteria and one of the most important industrial microorganisms due to superior biosynthetic capabilities of many valuable secondary metabolites as well as production of various valuable bioconversion enzymes. Among them are cytochrome P450 hydroxylase (CYP), which are hemoproteins encoded by a super family of genes, are universally distributed in most of the organisms from all biological kingdoms. Actinomycetes are a rich source of soluble CYP enzymes, which play critical roles in the bioactivation and detoxification of a wide variety of metabolite biosynthesis and xenobiotic transformation. Cyclosporin A (CyA), one of the most commonly-prescribed immunosuppressive drugs, was previously reported to be hydroxylated at the position of 4th N-methyl leucine by a rare actinomycetes called Sebekia benihana, leading to display different biological activity spectrum such as loss of immunosuppressive activities yet retaining hair growth-stimulating side effect. In order to improve this regio-selective CyA hydroxylation in S. benihana, previously-identified several secondary metabolite up-regulatory genes from Streptomyces coelicolor and S. avermitilis were heterologously overexpressed in S. benihana using an $ermE^*$ promoter-containing Streptomyces integrative expression vector. Among tested, SCO4967 encoding a conserved hypothetical protein significantly stimulated region-specific CyA hydroxylation in S. benihana, implying that some common regulatory systems functioning in both biosynthesis and bioconversion of secondary metabolite might be present in different actinomycetes species.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.890-901
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• 2021

The 11α-hydroxylation of canrenone can be catalyzed by Aspergillus ochraceus in bioreactors, where the geometry of the impeller greatly influences the biotransformation. In this study, the effects of the blade number and impeller diameter of a Rushton turbine on the 11α-hydroxylation of canrenone were considered. The results of fermentation experiments using a 50 mm four-blade impeller showed that 3.40% and 11.43% increases in the conversion ratio were achieved by increasing the blade number and impeller diameter, respectively. However, with an impeller diameter of 60 mm, the conversion ratio with a six-blade impeller was 14.42% lower than that with a four-blade impeller. Data from cold model experiments with a large-diameter six-blade impeller indicated that the serious leakage of inclusions and a 22.08% enzyme activity retention led to a low conversion ratio. Numerical simulations suggested that there was good gas distribution and high fluid flow velocity when the fluid was stirred by large-diameter impellers, resulting in a high dissolved oxygen content and good bulk circulation, which positively affected hyphal growth and metabolism. However, a large-diameter six-blade impeller created overly high shear compared to a large-diameter four-blade impeller, thereby decreasing the conversion ratio. The average shear rates of the former and latter cases were 43.25 s^-1 and 35.31 s^-1, respectively. We therefore concluded that appropriate shear should be applied in the 11α-hydroxylation of canrenone. Overall, this study provides basic data for the scaled-up production of 11α-hydroxycanrenone.

• Applied Biological Chemistry
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• v.17 no.3
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• pp.149-176
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• 1974

Eight substituted diphenyl ether herbicides and some of their photoproducts were studied in terms of solution phase photolysis under simulated environmental conditions by using a Rayonet photochemical reactor. The test compounds absorbed sufficient light energy at the wavelength of 300 nm to undergo various photoreactions. All the photoproducts were confirmed by means of tlc, glc, ir, ms, and/or nmr spectrometry. The results obtained are summarized as follows: Solution phase photolysis of C-6989: An exceedingly large amount of p-nitrophenol formed strongly indicates the readiness of the ether linkage cleavage of this compound as the main reaction in all solvents used. Photoreduction of nitro to amino group(s) and photooxidation of trifluoromethyl to carboxyl group were recognized as minor reactions. Aqueous photolysis of p-nitrophenol: Quinone(0.28%), hydroquinone (0.66%), and p-aminophenol (0.42%) were confirmed as photoproducts, in addition to a relatively small amount of an unknown compound. The mechanisms of formation of these products were proposed to be the nitro-nitrite rearrangement via $n{\rightarrow}{\pi}^*$ excitation and the photoreduction through hydrogen abstractions by radicals, respectively. Solution phase photolysis of Nitrofen: Photochemical reduction leading to the p-amino derivative was the main reaction in n-hexane. In aqueous solution, the photoreduction of nitro to amino group and hydroxylation predominated over the ether linkage cleavage. Nucleophilic displacement of the nitro group by hydroxide ion and replacement of chlorine substituents by hydroxyl group or, to a lesser extent, hydrogen were also observed as minor reactoins. Solution phase photolysis of MO-338: Photoreduction of the nitro to amino group was marked in the n-hexane solution photolysis. In the aqueous solution, photoreduction of the nitro substituent and hydroxylation were the main reactions with replacement of chlorine substituents by the hydroxyl group and hydrogen, and cleavage of the ether linkage as minor reactions. Photolyses of MC-4379, MC-3761, MC-5127, MC-6063, and MC-7181 in n-hexane and cyclohexane: Photoreduction of the nitro group leading to the corresponding amino derivative and replacement of one of the halogen substituents by hydrogen from the solvent used were the key reactions in each compound. Aqueous photolysis of MC-4379: Cleavage of the ether linkage, replacement of the carboxymethyl by hydroxyl group, hydroxylation, and replacement of the nitro by hydroxy group were prominent with photoreduction and dechlorination as minor reactions. Aqueous photolysis of MC-3761: Cleavage of the ether linkage, replacement of the carboxymethyl by hydroxyl group, and photoreduction followed by hydroxylation were the main reactions. Aqueous photolysis of MC-5127: Replacement of carboxyethyl by hydrogen was predominant with ether linkage cleavage, photoreduction, and dechlorination as minor reactions. It was obvious that the decarboxyethylation proceeded more readily than decarboxymethylation occurring in the other compounds. Aqueous photolysis of MC-6063: Cleavage of the ether linkage and photodechlorination were the main reactions. Aqueous photolysis of MC-7181: Replacement of the carboxymethyl group by hydrogen and monodechlorination were the remarkable reactions. Cleavage of the ether linkage and hydroxylation were thought to be the minor reactions. Aqueous photolysis of 3-carboxymethyl-4-nitrophenol: The photo-induced Fries rearrangement common to aromatic esters did not appear to occur in the carboxymethyl group of this type of compound. Conversion of nitro to nitroso group was the main reaction.

• Toxicological Research
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• v.11 no.1
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• pp.23-29
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• 1995

Microsomes from human liver sample HL 110 oxidized aflatoxin $B_1$ $(AFB_1)$ to $AFB_1$ exo-8, 9-epoxide which was detected as a glutathione (GSH) conjugate with excess GSH S-transferase and to aflatoxin $Q_1$ ($AFB_1$; 3$\alpha$-hydroxyafiatoxin $B_1$), and testosterone to 6$\beta$-hydroxytestosterone. Anti-P450 3A4 nearly completely inhibited all of the reactions. Some fiavonoids inhibited all of the reactions. While other fiayonolds stimulated 8, 9-epoxidation and inhibited 3$\alpha$-hydroxylation. Gestodene inhibited all of the reactions when gestodene was metabolized by human liver microsomal P450 3A4 prior to adding substrate. But, ges-todene was added in the enzyme mixtures in the presence of $AFB_1$, it could not inhibit 8, 9-epoxidation of $AFB_1$. Nifedipine and troleandomycin inhibited both of the reactions of $AFB_1$ but only 3$\alpha$-hydroxylation was inhibited by the oxidation product of nifedipine. Although, troleandomycin was known as a mechanism-based inhibitor, the chemical did not show any detectable inhibitory effect on 6$\beta$-hydroxylation of testosterone. The results suggest that there are several different substrate-binding sites on P450 3A4.