- A radiometric assay method for aromatase activity using [1β-3H]16α-hydroxyandrostenedione
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[1β-3H]16α-Hydroxyandrostenedione (16α-OHA) (715 mCi/mmol) was prepared from commercially available [1β-3H]androstenedione (A) by the microbiological method with Streptomyces roseochromogenes and its structure and purity were determined by chromatographic and reverse isotope dilution methods. When [1β-3H]16α-OHA was incubated with human placental microsomes and reduced nicotinamide adenine dinucleotide phosphate (NADPH), 3H2O-release into the medium was dependent upon protein concentration and incubation time. An apparent K(m) and V(max) of the microsomal aromatase for the [1β-3H]substrate were 650 nM and 34 pmol/min/mg protein, respectively. In this assay, aromatase activity could be determined as low as 0.1 nmol estrogen formation/min/mg protein. 3-Deoxyandrostenedione, a potent competitive inhibitor of the A aromatization, also blocked the 16α-OHA aromatization in a competitive manner with K(i) of 15 nM.
- Numazawa,Mutsumi,Nakakoshi,Nagaoka
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- CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**
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CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.
- Bracco, Paula,Wijma, Hein J.,Nicolai, Bastian,Buitrago, Jhon Alexander Rodriguez,Klünemann, Thomas,Vila, Agustina,Schrepfer, Patrick,Blankenfeldt, Wulf,Janssen, Dick B.,Schallmey, Anett
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p. 1099 - 1110
(2020/12/03)
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- Oxidative Diversification of Steroids by Nature-Inspired Scanning Glycine Mutagenesis of P450BM3 (CYP102A1)
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Steroidal compounds are some of the most prescribed medicines, being indicated for the treatment of a variety of conditions including inflammation, heart disease, and cancer. Synthetic approaches to functionalized steroids are important for generating steroidal agents for drug screening and development. However, chemical activation is challenging because of the predominance of inert, aliphatic C-H bonds in steroids. Here, we report the engineering of the stable, highly active bacterial cytochrome P450 enzyme P450BM3 (CYP102A1) from Bacillus megaterium for the mono- and dihydroxylation of androstenedione (AD), dehydroepiandrosterone (DHEA), and testosterone (TST). In order to design altered steroid binding orientations, we compared the structure of wild type P450BM3 with the steroid C19-demethylase CYP19A1 with AD bound within its active site and identified regions of the I helix and the β4 strand that blocked this binding orientation in P450BM3. Scanning glycine mutagenesis across 11 residues in these two regions led to steroid oxidation products not previously reported for P450BM3. Combining these glycine mutations in a second round of mutagenesis led to a small library of P450BM3 variants capable of selective (up to 97%) oxidation of AD, DHEA, and TST at the widest range of positions (C1, C2, C6, C7, C15, and C16) by a bacterial P450 enzyme. Computational docking of these steroids into molecular dynamics simulated structures of selective P450BM3 variants suggested crucial roles of glycine mutations in enabling different binding orientations from the wild type, including one that closely resembled that of AD in CYP19A1, while other mutations fine-tuned the product selectivity. This approach of designing mutations by taking inspiration from nature can be applied to other substrates and enzymes for the synthesis of natural products and their derivatives.
- Cao, Yang,Chen, Wenyu,Fisher, Matthew J.,Leung, Aaron,Wong, Luet L.
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p. 8334 - 8343
(2020/09/18)
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- Tracking Down a New Steroid-Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233-SM
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CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11-ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α-hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16-hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α-dihydroxyprogesterone and 6β,16α-dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications.
- Dangi, Bikash,Kim, Ki-Hwa,Kang, Sang-Ho,Oh, Tae-Jin
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p. 1066 - 1077
(2018/04/30)
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- Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution
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CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO3+), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H2O2, with optimum conversion surprisingly being achieved at ≈75 mm H2O2. More importantly, H2O2 tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H2O2. Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.
- Dangi, Bikash,Park, Hyun,Oh, Tae-Jin
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p. 2273 - 2282
(2018/10/20)
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- Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: Substrate specificity and inhibitor selectivity studies
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Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring α-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363 → Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wildtype. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L- threo-chloramphenicol was abolished in the Val-367 → Ala mutant. The Val- 363 → Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367 → Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.
- You Qun He,You Ai He,Halpert
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p. 574 - 579
(2007/10/03)
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- Stereoselective hydrolysis of 16α-halo-17-keto steroids and long-range substitution effects on the hydrolysis of 16α-bromo-17-ketones and 2α-bromo-3-ketones
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Epimerization of 16α-chloro- (1a), bromo- (1b), and iodo-3β-hydroxy-5-androsten-17-one (1c) by a brief treatment with 0.2 equiv NaOH in aqueous pyridine reached equilibrium between 16α- and 16β-halo ketones. 16α-/16β-Halo ketone ratios at equilibrium were 1.5 for Cl, 1.25 for Br, and 1.0 for I. Kinetic analysis showed that compounds 1a-c were stereoselectively converted to the corresponding 16α-hydroxy derivative 3 by an S(N)2 mechanism, in which the order of the apparent reactivity was Br > I > Cl. The hydrolysis of a number of 16α-bromo-17-ketones and 2α-bromo-3-ketones was carried out. The yields of the corresponding alcohols were found to depend on remote structural features in the steroids.
- Numazawa,Ogata,Abiko,Nagaoka
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p. 403 - 410
(2007/10/02)
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- Stereospecific Synthesis of 16α-Hydroxy-17-oxo Steroids by Controlled Alkaline Hydrolysis of Corresponding 16-Bromo 17-Ketones and Its Reaction Mechanism
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Synthesis of 16α-hydroxy-17-oxo steroids 3, 5b, and 3β,16α-dihydroxy-5-17-oxoandrosten-3-yl sulfate (7) from 16α-bromo-17-oxo steroids 1, 5a, and 6a and the reaction mechanism of the controlled alkaline hydrolysis are described.Treatment of the bromo ketones with NaOH in aqueous DMF gave the 16α-hydroxy 17-ketones stereoselectively in 95percent yield without formation of other ketols.The sodium salt of 3-sulfate 7 was also obtained in one step in 85percent yield from the corresponding bromo ketone (1a).Isotope-labeling experiments and time-course studies showed that equilibration between the 16-bromo epimers 1 and 2 precedes the formation of 3, in which the true intermediate is 2 and not 1, and that the ketol 3 is formed by the direct SN2 displacement of the 16β-bromine.The 16β-morpholino derivative 8 obtained by reaction of 1 with morpholine was shown to be an isomerized product of the 16α isomer which is produced also by SN2 displacement of the 16β-bromine.The mechanism of ketol rearrangement of 3 to the 17β-hydroxy-16-oxo compound 4 was found to involve a hydration to the carbonyl function.The new hydration-dehydration mechanism is proposed for the ketol rearrangement.
- Numazawa, Mitsuteru,Nagaoka, Masao
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p. 4024 - 4029
(2007/10/02)
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- CONTROLLED ALKALINE HYDROLYSIS OF STEROIDAL α-BROMOKETONES: NEW CONDITIONS AND SYNTHESIS OF 2α-HYDROXY-3-ONES
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Controlled alkaline hydrolysis of 16α-bromo-17-keto steroids 1, 5 and 7 with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu4NOH) and synthesis of 2α-hydroxy-3-ones 11, 13 and 16 by the controlled hydrolysis of the corresponding 2α-bromo-3-ones 9, 12 and 15 are described.Treatment of the bromoketones 1, 5 and 7 with potassium carbonate in aqueous acetone or with n-Bu4NOH in aqueous dimethylformamide (DMF) gave 16α-hydroxy-17-ones 3, 6 and 8 in 85-90percent yield, respectively. 2α-Hydroxy-3-ones 11, 13 and 16 were obtained by hydrolysis of the corresponding bromoketones 9, 12 and 15 in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively.Deuterium labeling experiments suggested that equilibration between the 2α-bromoketone 9 and the 2β-bromo isomer 10 precedes the formation of the ketol 11 in which the true intermediate might be the 2β-isomer 10.However, rearranged androstane derivatives, 3β-hydroxy-2-ones 18 and 20, were stereoselectively obtained by treatment of the bromoketones 12 and 15 with an excess amount of sodium hydroxide.
- Numazawa, Mitsuteru,Nagaoka, Masao
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p. 345 - 356
(2007/10/02)
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- SYNTHESIS OF 16α-BROMOACETOXY ANDROGENS AND 17β-BROMOACETYLAMINO-4-ANDROSTEN-3-ONE: POTENTIAL AFFINITY LABELS OF HUMAN PLACENTAL ARMATASE.
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A novel synthesis of 16α-hydroxy-4-androstene-3,17-dione (3), 16α-hydroxy-4-androstene-3,6,17-trione (4), 17β-amino-5-androsten-3β-ol (10) and 17β-amino-4-androsten-3-one (14) is described. 16α-Bromoacetoxy-4-androstene-3,17-dione (5), 16α-bromoacetoxy-4-androstene-3,6,17-trione (6) and 17β-bromoacetylamino-4-androsten-3-one (15) were synthesized as potentially selective irreversible inhibitors of androgen aromatases. 16α-Bromo-4-androstene-3,17-dione (1) and 16α-bromo-4-androstene-3,6,17-trione (2) were converted to compounds 3 and 4 in 80-90percent yield by controlled stereospecific hydrolysis using sodium hydroxide in aqueous pyridine.Reductive amination of 3β-hydroxy-5-androsten-17-one and 3-methoxy-3,5-androstadien-17-one (11) using ammonium acetate and sodium cyanohydridoborate (NaBH3CN) and a subsequent treatment with acid gave the amines 10 and 14, respectively, as a salt.The corresponding 17-imino compounds 9 and 13 were also isolated from the reaction mixtures when methanol was used as a solvent for the reaction.The 16α-hydroxyl compounds 3 and 4 and the 17β-amino compound 14 were converted to the corresponding bromoacetyl derivatives, 5, 6, and 15, with bromoacetic acid and N,N'-dicyclohexylcarbodiimide.
- Numazawa, Mitsuteru,Osawa, Yoshio
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p. 149 - 160
(2007/10/02)
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