• 약학회지
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• 제41권5호
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• pp.638-646
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• 1997

The steroid 9${alpha}$-hydroxylase activity has been detected in cytosol fraction, $100,00{\times}g$ supernatant of cell free extract of Mycobacterium fortuitum. The activity was not linear with protein concentration in the assay suggesting 9${alpha}$-hydroxylase is a multicomponent enzyme. The 9${alpha}$-hydroxylase system was partially purified through fractional saturation of ammonium sulfate, strong anion exchange (Mono Q) column chromatography, gel filtration (Superose 12) column chromatography, and testosterone affinity gel chromatography. Ammonium sulfate 50~60% saturated fraction of the cytosol gave 9${alpha}$-hydroxylase activity. For further purification, the half-saturated ammonium sulfate fraction was applied to Mono Q, Superose 12, or affinity gel column. The purification factors of 9${alpha}$-hydroxylase containing fraction after Mono Q, Superose 12, and affinity gel chromatography was 13, 11, and 17 respectively.

• Archives of Pharmacal Research
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• 제20권6호
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• pp.519-524
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• 1997

Steroid $9{\alpha}$-hydroxylase is an enzyme found in nocardioform microorganisms which can utilize steroids as a sole carbon source. After fractional centrifugation of the cell homogenates, the enzyme activity in Nocardia and Rhodococcus was found in cytoplasmic membrane fraction. On the contrary, Mycobacterium had its 9.alpha.-hydroxylation activity in cytosolic fraction. To characterize the enzyme in these microorganisms, several potential inhibitors of 9.alpha.-hydroxylase were tested and the cofactor requirement for the same enzyme was also examined. The inhibitory effect of ferrous ion chelators indicated involvement of iron containing proteins in the 9.alpha.-hydroxylase system. On the other hand, metyrapone, an inhibitor known to be specific for cytochrome P450 interfered with the enzyme in Mycobacterium, but didn't inhibit the enzyme activity in Nocardia and Rhodococcus. While the $9{\alpha}$-hydroxylase system in Nocardia and Rhodococcus required NADPH, NADH was required as an election donor in Mycobacterium.

• Archives of Pharmacal Research
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• 제20권6호
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• pp.590-596
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• 1997

The NADH reductase component of the steroid 9.alpha.-hydroxylase from Mycobacterium fortuitum was purified to homogeneity. Recovery of the enzyme from the 50-60% ammonium sulfate saturated fraction was 49%, with a purification factor of 100-fold. The NADH reductase has a relative molecular of 60 KDa as determined by SDS-PAGE. The absorption maxima at 410 and 450 nm indicate the presence of iron-sulfur group and flavin. These prosthetic groups seemed to function as redox groups that transfer electrons from NADH to the following protein. The $K_M$ value for NADH as substrate was $68{\mu}M$. The $NH_2$-terminal amino acid sequence of the reductase was determined as Met-Asp-Ala-Ile-Thr-Asn-Val-Pro-Leu-Pro-Ala-Asn-Glu-Pro-Val-His-Asp-Tyr-Ala-Thr. This sequence does not show a homology with the $NH_2$ -terminal sequences reported for the reductase component of other monooxygenases, suggesting that the NADH reductase component of the steroid 9.alpha.-hydroxylase system is novel.

• Applied Biological Chemistry
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• 제27권2호
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• pp.100-106
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• 1984

신생 쥐 간의 subcellular fraction의 특징과 $6{\alpha}$-steroid hydroxylase의 subcellular localization및 특성을 살펴본 결과는 다음과 같다. 1000g 30초 침전 fraction은 총 DNA의 95%를 차지하므로써 crude nuclei fraction으로 나타났으며 또한 5'-nucleotidase의 specific activity가 높은 것은 파괴되지 않은 세포에 기인된 것으로 생각이 되며, 1450g 10분 침전 fraction은 5'-nucleotidase의 activity가 가장 높으므로 crude plasma membrane fraction으로 동정하였으나 mitochondria가 같이 침전되어 있었으며, 9000g 20분 침전 fraction은 succinate-cytochrome C reductase의 activity가 가장 높아 mitochondria fraction으로 하였고, 105,000g 60분 상등액은 LDH가 가장 높아 cytosol로 하였으며, 105,000g 60분 침전은 남은 부분으로 microsome fraction으로 추정하였다. 각 fraction에 기질인 $3{\beta}$-hydroxy-$5{\alpha}$-pregnan-20-one을 incubation시킨 후의 생성된 steroid를 보면 crude nuclei fraction에서는 pregnadiol($5{\alpha}$-pregnane-$3{\alpha}/{\beta}$, $20{\alpha}$-diol)의 형성으로 $20{\alpha}$-reduction과 $3{\alpha}$-$3{\beta}$ iso${\beta}$merization을 볼 수 있었으며 crude plasma membrane fraction에서는 $20{\alpha}$-reduction과 $6{\alpha}$-hydroxylation을 볼 수 있었으며 crude mitochondria와 cytosol에서는 $20{\alpha}$-reduction만을 crude microsome에서는 $16{\alpha}$-hydroxylation을 볼 수 있었으므로 $6{\alpha}$-hydroxylase는 crude plasma membrane에 존재함을 확인하였다. 한편 $6{\alpha}$-steroid hydroxylase에 존재함을 확인하였다. 한편 $6{\alpha}$-steroid hydroxylase의 최대 활성도는 pH 7에서 나타났으며 progesterone은 hydroxylation을 시키지 못한 반면 $3{\alpha}$-hydroxy-$5{\alpha}$-pregnan-20-one은 $6{\alpha}$-hydroxylation이 되었다.

• Archives of Pharmacal Research
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• 제20권6호
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• pp.525-528
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• 1997

Steroid $9{\alpha}$.-hydroxylase is a key enzyme system in steroid nucleus degradation in company with ${\Delta}$-dehydrogenase. To examine $9{\alpha}$-hydroxylase activity during microbial transformation of steroids, 9(11)-dehydro-$17{\alpha}$-methyl-testosterone was adopted as a stable substrate for preventing the rupture of steroid nucleus. Using Nocardia restrictus ATCC 14887 capable of introducing a $9{\alpha}$-hydroxyl group into steroids, $9{\alpha}$,$11{\alpha}$-oxido-$17{\beta}$-hydroxy-$17{\alpha}$-methyl-4-androstene-3-one and $9{\alpha}$-hydroxyl group into steroids,$9{\alpha}$,$11{\alpha}$-oxido-$17{\beta}$-hydroxy-$17{\alpha}$-methyl-1,4-androstadiene-3- one were obtained. These microbiologically transformed products could be used as reference compounds in the enzyme assay.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.387-397
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• 2023

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1472-1482
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• 2017

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity ($K_d$ value) to 4-androstenedione was $387{\pm}37{\mu}M$. Moreover, the crystal structure of BaCYP106A2 was determined at $2.7{\AA}$ resolution. Structural analysis suggested that the ${\alpha}8-{\alpha}9$ loop region of BaCYP106A2 is intrinsically mobile and might be important for initial ligand binding. The hydroxyl activity of BaCYP106A2 was identified using in vitro enzyme assays. Its activity was confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using chromatography and mass spectrometry methods. The main products were mono-hydroxylated compounds with high conversion yields. This is the second study on the structure of CYP106A steroid hydroxylases, and should contribute new insight into the interactions of bacterial CYP106A with steroid substrates, providing baseline data for studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

• 미생물학회지
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• 제27권4호
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• pp.366-372
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• 1989

Reichstein's substance S의 $11\beta$-hydroxylation 활성이 있는 균주를 선택하고자 21 균주를 실험한 결과 4균주에서 $11\beta$-hydroxylation의 활성이 발견되었으며, 그 중 Pellicularia fillamentosark 가장 높은 효소활성을 보였다. 이 균주의 $11\beta$-hydroxylation의 합성을 강력하게 저해하였다. $11\beta$-hydroxylation의 최적 pH 범위는 2.0-8.0으로 광범위하였다. 성장단계 중 균사체 분지시기와 완전하게 성숙한 균사체 시기에 효소합성의 유도가 가장 활발하였으며, 70시간 배양된 균사체가 가장 좋았다. 반면, 포자에는 기질에 의한 $11\beta$-hydroxylation의 유도현상이 없는 것으로 판정되었다.

• Clinical and Experimental Pediatrics
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• 제51권9호
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• pp.1018-1022
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• 2008

선천 부신 과다형성 환자에서 고환 부신 잔류 종양은 흔하게 발생한다. 대개 이 종양은 적절한 corticosteroid 억제 치료로 호전될 수 있다. 저자들은 양측성 고환 부신 잔류 종양을 보인 21-hydroxylase 결핍증 환아에게서 corticosteroid를 투여하였으나 반응하지 않아 고환 적출술을 시행한 사례를 경험하였기에 보고하는 바이다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2007년도 Proceedings of The Convention
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• pp.57-64
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• 2007

Metabolic interactions of the three major cannabinoids, ${\Delta}^9$-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) with steroid hormones were investigated. These cannabioids concentration-dependently inhibited $3{\beta}$-hydroxysteroid dehydrogenase and $17{\alpha}$-hydroxylase in rat adrenal and testis microsomes. CBD and CBN were the most potent inhibitors of $3{\beta}$-phydroxysteroid dehydrogenase and progesterone $17{\alpha}$-hydroxylase, respectively, in rat testis microsomes. Three cannabinoids highly attenuated hCG-stimulated testosterone production in rat testicular interstitial cells. These cannabinoids also decreased in levels of mRNA and protein of StAR in the rat testis cells. These results indicate that the cannabinoids could interact with steroid hormones, and exert their modulatory effects on endocrine and testicular functions. Metabolic interaction of a THC metabolite, $7{\beta}$-hydroxy-${\Delta}^8$-THC with steroids is also investigated. Monkey liver microsomes catalyzed the stereoselective oxidation of $7{\beta}$-hydroxy-${\Delta}^8$-THC to 7-oxo-${\Delta}^8$-THC, so-called microsomal alcohol oxygenase (MALCO). The reaction is catalyzed by CYP3A8 in the monkey liver microsomes, and required NADH as well as NADPH as an efficient cofactor, and its activity is stimulated by some steroids such as testosterone and progesterone. Kinetic analyses revealed that MALCO-catalyze reaction showed positive cooperativity. In order to explain the metabolic interaction between the cannabinoid metabolite and testosterone, we propose a novel kinetic model involving at least three binding sites for mechanism of the metabolic interactions.