1.Functional Characterization of Pharmcogenetic Variants of Human Cytochrome P450 2C9 in Korean Populations
Myung A CHO ; Jihoon G YOON ; Vitchan KIM ; Harim KIM ; Rowoon LEE ; Min Goo LEE ; Donghak KIM
Biomolecules & Therapeutics 2019;27(6):577-583
Human cytochrome P450 2C9 is a highly polymorphic enzyme that is required for drug and xenobiotic metabolism. Here, we studied eleven P450 2C9 genetic variants—including three novel variants F69S, L310V, and Q324X—that were clinically identified in Korean patients. P450 2C9 variant enzymes were expressed in Escherichia coli and their bicistronic membrane fractions were prepared The CO-binding spectra were obtained for nine enzyme variants, indicating P450 holoenzymes, but not for the M02 (L90P) variant. The M11 (Q324X) variant could not be expressed due to an early nonsense mutation. LC-MS/MS analysis was performed to measure the catalytic activities of the P450 2C9 variants, using diclofenac as a substrate. Steady-state kinetic analysis revealed that the catalytic efficiency of all nine P450 2C9 variants was lower than that of the wild type P450 2C9 enzyme. The M05 (R150L) and M06 (P279T) variants showed high k(cat) values; however, their K(m) values were also high. As the M01 (F69S), M03 (R124Q), M04 (R125H), M08 (I359L), M09 (I359T), and M10 (A477T) variants exhibited higher K(m) and lower k(cat) values than that of the wild type enzyme, their catalytic efficiency decreased by approximately 50-fold compared to the wild type enzyme. Furthermore, the novel variant M07 (L310V) showed lower k(cat) and K(m) values than the wild type enzyme, which resulted in its decreased (80%) catalytic efficiency. The X-ray crystal structure of P450 2C9 revealed the presence of mutations in the residues surrounding the substrate-binding cavity. Functional characterization of these genetic variants can help understand the pharmacogenetic outcomes.
Codon, Nonsense
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Cytochrome P-450 Enzyme System
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Cytochromes
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Diclofenac
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Escherichia coli
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Holoenzymes
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Humans
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Membranes
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Metabolism
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Pharmacogenetics
2.Streptomyces Cytochrome P450 Enzymes and Their Roles in the Biosynthesis of Macrolide Therapeutic Agents
Myung A CHO ; Songhee HAN ; Young Ran LIM ; Vitchan KIM ; Harim KIM ; Donghak KIM
Biomolecules & Therapeutics 2019;27(2):127-133
The study of the genus Streptomyces is of particular interest because it produces a wide array of clinically important bioactive molecules. The genomic sequencing of many Streptomyces species has revealed unusually large numbers of cytochrome P450 genes, which are involved in the biosynthesis of secondary metabolites. Many macrolide biosynthetic pathways are catalyzed by a series of enzymes in gene clusters including polyketide and non-ribosomal peptide synthesis. In general, Streptomyces P450 enzymes accelerate the final, post-polyketide synthesis steps to enhance the structural architecture of macrolide chemistry. In this review, we discuss the major Streptomyces P450 enzymes research focused on the biosynthetic processing of macrolide therapeutic agents, with an emphasis on their biochemical mechanisms and structural insights.
3.Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11.
Seunghye CHOI ; Songhee HAN ; Hwayoun LEE ; Young Jin CHUN ; Donghak KIM
Biomolecules & Therapeutics 2013;21(6):487-492
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 K m. 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.
Animals
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Arachidonic Acid
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Blood Pressure
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Cats
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Clinical Coding
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Cytochrome P-450 Enzyme System
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Escherichia coli
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Fatty Acids, Unsaturated
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Humans*
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Hydroxylation
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Liver
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Luciferases
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Luminescence
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Mass Screening
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Mutagenesis
4.Structural Insights into the Interaction of Terpenoids with Streptomyces avermitilis CYP107P2
Eunseo JEONG ; Vitchan KIM ; Changmin KIM ; Yoo-bin LEE ; Donghak KIM
Biomolecules & Therapeutics 2024;32(4):474-480
Streptomyces avermitilis genome includes 33 genes encoding monooxygenation-catalyzing cytochrome P450 enzymes. We investigated the structure of CYP107P2 and its interactions with terpenoid compounds. The recombinant CYP107P2 protein was expressed in Escherichia coli and the purified enzyme exhibited a typical P450 spectrum upon CO-binding in its reduced state. Type-I substrate-binding spectral titrations were observed with various terpenoid compounds, including α-pinene, β-pinene, α-terpinyl acetate, and (+)-3-carene. The calculated binding affinities (Kd) ranged from 15.9 to 50.8 µM. The X-ray crystal structure of CYP107P2 was determined at 1.99 Å resolution, with a well-conserved overall P450 folding conformation. The terpenoid com-pound docking models illustrated that the structural interaction between monoterpenes and CYP107P2, with the distance between heme and terpenes ranging from 3.4 to 5.4 Å, indicates potential substrate binding for P450 enzyme. This study suggests that CYP107P2 is a Streptomyces P450 enzyme capable of catalyzing terpenes as substrates, signifying noteworthy advancements in comprehending a novel P450 enzyme’s involvement in terpene reactions.
5.Structural Insights into the Interaction of Terpenoids with Streptomyces avermitilis CYP107P2
Eunseo JEONG ; Vitchan KIM ; Changmin KIM ; Yoo-bin LEE ; Donghak KIM
Biomolecules & Therapeutics 2024;32(4):474-480
Streptomyces avermitilis genome includes 33 genes encoding monooxygenation-catalyzing cytochrome P450 enzymes. We investigated the structure of CYP107P2 and its interactions with terpenoid compounds. The recombinant CYP107P2 protein was expressed in Escherichia coli and the purified enzyme exhibited a typical P450 spectrum upon CO-binding in its reduced state. Type-I substrate-binding spectral titrations were observed with various terpenoid compounds, including α-pinene, β-pinene, α-terpinyl acetate, and (+)-3-carene. The calculated binding affinities (Kd) ranged from 15.9 to 50.8 µM. The X-ray crystal structure of CYP107P2 was determined at 1.99 Å resolution, with a well-conserved overall P450 folding conformation. The terpenoid com-pound docking models illustrated that the structural interaction between monoterpenes and CYP107P2, with the distance between heme and terpenes ranging from 3.4 to 5.4 Å, indicates potential substrate binding for P450 enzyme. This study suggests that CYP107P2 is a Streptomyces P450 enzyme capable of catalyzing terpenes as substrates, signifying noteworthy advancements in comprehending a novel P450 enzyme’s involvement in terpene reactions.
6.Structural Insights into the Interaction of Terpenoids with Streptomyces avermitilis CYP107P2
Eunseo JEONG ; Vitchan KIM ; Changmin KIM ; Yoo-bin LEE ; Donghak KIM
Biomolecules & Therapeutics 2024;32(4):474-480
Streptomyces avermitilis genome includes 33 genes encoding monooxygenation-catalyzing cytochrome P450 enzymes. We investigated the structure of CYP107P2 and its interactions with terpenoid compounds. The recombinant CYP107P2 protein was expressed in Escherichia coli and the purified enzyme exhibited a typical P450 spectrum upon CO-binding in its reduced state. Type-I substrate-binding spectral titrations were observed with various terpenoid compounds, including α-pinene, β-pinene, α-terpinyl acetate, and (+)-3-carene. The calculated binding affinities (Kd) ranged from 15.9 to 50.8 µM. The X-ray crystal structure of CYP107P2 was determined at 1.99 Å resolution, with a well-conserved overall P450 folding conformation. The terpenoid com-pound docking models illustrated that the structural interaction between monoterpenes and CYP107P2, with the distance between heme and terpenes ranging from 3.4 to 5.4 Å, indicates potential substrate binding for P450 enzyme. This study suggests that CYP107P2 is a Streptomyces P450 enzyme capable of catalyzing terpenes as substrates, signifying noteworthy advancements in comprehending a novel P450 enzyme’s involvement in terpene reactions.
7.Characterization of a Biflaviolin Synthase CYP158A3 from Streptomyces avermitilis and Its Role in the Biosynthesis of Secondary Metabolites.
Young Ran LIM ; Songhee HAN ; Joo Hwan KIM ; Hyoung Goo PARK ; Ga Young LEE ; Thien Kim LE ; Chul Ho YUN ; Donghak KIM
Biomolecules & Therapeutics 2017;25(2):171-176
Streptomyces avermitilis produces clinically useful drugs such as avermectins and oligomycins. Its genome contains approximately 33 cytochrome P450 genes and they seem to play important roles in the biosynthesis of many secondary metabolites. The SAV_7130 gene from S. avermitilis encodes CYP158A3. The amino acid sequence of this enzyme has high similarity with that of CYP158A2, a biflaviolin synthase from S. coelicolor A3(2). Recombinant S. avermitilis CYP158A3 was heterologously expressed and purified. It exhibited the typical P450 Soret peak at 447 nm in the reduced CO-bound form. Type I binding spectral changes were observed when CYP158A3 was titrated with myristic acid; however, no oxidative product was formed. An analog of flaviolin, 2-hydroxynaphthoquinone (2-OH NQ) displayed similar type I binding upon titration with purified CYP158A3. It underwent an enzymatic reaction forming dimerized product. A homology model of CYP158A3 was superimposed with the structure of CYP158A2, and the majority of structural elements aligned. These results suggest that CYP158A3 might be an orthologue of biflaviolin synthase, catalyzing C-C coupling reactions during pigment biosynthesis in S. avermitilis.
Amino Acid Sequence
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Cytochrome P-450 Enzyme System
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Genome
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Myristic Acid
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Oligomycins
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Streptomyces*
8.Functional Significance of Cytochrome P450 1A2 Allelic Variants, P450 1A2*8, *15, and *16 (R456H, P42R, and R377Q).
Young Ran LIM ; In Hyeok KIM ; Songhee HAN ; Hyoung Goo PARK ; Mi Jung KO ; Young Jin CHUN ; Chul Ho YUN ; Donghak KIM
Biomolecules & Therapeutics 2015;23(2):189-194
P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of approximately 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers (k(cat)) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency (k(cat)/K(m)) increased up to 2.5 fold with a slight increase of its K(m) value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.
Biotransformation
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Blotting, Western
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Cytochrome P-450 CYP1A2*
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Escherichia coli
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Genetic Variation
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Metabolism
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Phenacetin
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Polymorphism, Single Nucleotide
9.Optimal Culture Condition for Antifungal Susceptibility Tests of Malassezia globosa.
Beom Joon KIM ; Eun Chang LEE ; Yun Young LIM ; Donghak KIM ; Young Jin CHUN
Korean Journal of Medical Mycology 2009;14(4):182-189
BACKGROUND: Although numerous culture conditions for Malassezia species were suggested, there were not so many objective evaluation articles in the literature. OBJECTIVES: We examined the various culture conditions for Malassezia globosa. METHODS: Malassezia globosa culture conditions were assessed by Dixon's agar, modified Leeming-Notman medium in diverse oil content and temperature conditions. RESULTS: Maximum growth rate of Malassezia globosa was achieved at 3% olive oil. The optimal temperatures for the maximal growth of M. globosa were observed at 32~34degrees C. CONCLUSION: In this study, we established the optimal culture condition for M. globosa, and confirmed its excellent utility for the antifungal susceptibility tests for M. globosa and M. restricta. Our results can help the investigators plan to do the prospective researches involving Malassezia species, such as the susceptibility test for newly developed antifungal agents.
Agar
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Antifungal Agents
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Humans
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Malassezia
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Olea
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Olive Oil
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Plant Oils
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Research Personnel
10.Validity and reliability of the Korean version of the Quality of Recovery-40 questionnaire.
Jun Ho LEE ; Deokkyu KIM ; Donghak SEO ; Ji seon SON ; Dong Chan KIM
Korean Journal of Anesthesiology 2018;71(6):467-475
BACKGROUND: The Quality of Recovery-40 (QoR-40) is a widely-used, self-rated, and self-completed questionnaire for postoperative patients. The questionnaire is intended to elicit information from each patient regarding the quality of recovery during the postoperative period. It is noteworthy, however, that an official Korean version of the QoR-40 (QoR-40K) has not been established. The purpose of this study was to develop the QoR-40K by translation and cultural adaptation process and to evaluate the validity and reliability of the QoR-40K. METHODS: After pre-authorization from the original author of the QoR-40, the translation procedure was established and carried out based upon Beaton’s recommendation to create a QoR-40K model comparable to the original English QoR-40. Two hundred surgical patients were enrolled, and each completed the questionnaire during the preoperative period, on the third day, and 1 month after surgery. The QoR-40K was compared with the visual analogue scale (VAS) and another health-related questionnaire, the Short-form Health Survery-36 (SF-36). The method of validation for QoR-40K included test-retest reliability, internal consistency, and level of responsiveness. RESULTS: Spearman’s correlation coefficient for test-retest reliability was 0.895 (P < 0.001), and Cronbach’s alpha of the global QoR-40K on the third day after surgery was 0.956. A positive correlation was obtained between the QoR-40K and the mental component summary of SF-36 (ρ = 0.474, P < 0.001), and a negative correlation was observed between QoR-40K and VAS (ρ = −0.341, P < 0.001). The standardized responsive mean of the total QoR-40K was 0.71. CONCLUSIONS: The QoR-40K was found to be as acceptable and reliable as the original English QoR-40 for Korean patients after surgery, despite the apparent differences in the respective patients’ cultural backgrounds.
Cross-Cultural Comparison
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Humans
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Methods
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Postoperative Period
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Preoperative Period
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Quality of Life
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Reproducibility of Results*