- Biotransformation of methyltestosterone by the filamentous fungus Mucor racemosus
-
Fungi have proved to be powerful biocatalysts in steroid biotransformations. In the present study, the soil isolate filamentous fungus Mucor racemosus was applied for bioconversion of methyltestosterone (1), an anabolic steroid, in a five-day fermentation. Microbial metabolites were purified chromatographically and identified on the basis of their spectral data as 7α-hydroxymethyltestosterone (2), 15α-hydroxymethyltestosterone (3), and 12,15α-dihydroxymethyltestosterone (4). Observed modifications were hydroxylations at C-7α, C-12, and 15α-positions. Best fermentation condition for production of hydroxylated derivatives was found to be 25°C at 150 rpm for 5 days with a substrate concentration of 1 mg/mL.
- Torshabi,Badiee,Faramarzi,Rastegar,Forootanfar,Mohit
-
-
Read Online
- Efficient hydroxylation of functionalized steroids by Colletotrichum lini ST-1
-
Abstract Biotransformation of a series of steroid compounds (estradiol, estrone, androst-4-en-3,17-dione, testosterone, canrenone, 16α,17α-epoxyprogesterone and progesterone) with Colletotrichum lini ST-1 as biocatalyst was investigated. With the exception of estradiol, estrone and progesterone, the microorganism could selectively hydroxylate steroid substrates (4 g/L) with 70-85% conversion rate and 60-76% total products yield. The different hydroxylation sites between androst-4-en-3,17-dione (3) and testosterone (4) suggested that the hydroxyl group or carbonyl group on the substrate at C17 had profound influence on the location of introduced hydroxyl groups. Transformations of 3-keto-steroid (3, 4, 5, 6 and 7) included monohydroxylation or dihydroxylation at 11α and 15α positions, while hydroxylations of 3-hydroxy-steroid (DHEA) were hydroxylation at 7α and 15α positions. Moreover, time course experiments demonstrated dihydroxylation of androst-4-en-3,17-dione (3), canrenone (5) and 16α,17α-epoxyprogesterone (6) were all initiated by hydroxylation on ring-D (C15) followed by attack on ring-C (C11). In this study, several new hydroxylation products were discovered, including 11α,15α-dihydroxyandrost-4-en-3,17-dione (9), 11α,15α-dihydroxy-canrenone (12) and 11α,15α-dihydroxy-16α,17α-epoxyprogesterone (14). The breadth of substrate spectrum and the excellent conversion rates achieved with this fungus indicated that C. lini ST-1 was a potential microorganism for production of valuable pharmaceutical ingredients and precursors.
- Wu, Yan,Li, Hui,Zhang, Xiao-Mei,Gong, Jin-Song,Rao, Zhi-Ming,Shi, Jin-Song,Zhang, Xiao-Juan,Xu, Zheng-Hong
-
-
Read Online
- A Modified Arrhenius Approach to Thermodynamically Study Regioselectivity in Cytochrome P450-Catalyzed Substrate Conversion
-
The regio- (and stereo-)selectivity and specific activity of cytochrome P450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrate relative to the heme, and the activation barrier of the regioselective oxidation reaction(s). The accessibility of potential SOMs depends on the relative binding free energy (ΔΔGbind) of the catalytically active substrate-binding poses, and the probability of the substrate to adopt a transition-state geometry. An established experimental method to measure activation energies of enzymatic reactions is the analysis of reaction rate constants at different temperatures and the construction of Arrhenius plots. This is a challenge for multistep P450-catalyzed processes that involve redox partners. We introduce a modified Arrhenius approach to overcome the limitations in studying P450 selectivity, which can be applied in multiproduct enzyme catalysis. Our approach gives combined information on relative activation energies, ΔΔGbind values, and collision entropies, yielding direct insight into the basis of selectivity in substrate conversion.
- Luirink, Rosa A.,Verkade-Vreeker, Marlies C. A.,Commandeur, Jan N. M.,Geerke, Daan P.
-
p. 1461 - 1472
(2020/03/03)
-
- Preparative-Scale Production of Testosterone Metabolites by Human Liver Cytochrome P450 Enzyme 3A4
-
Just like the drugs themselves, their metabolites have to be evaluated to succeed in a drug development and approval process. It is therefore essential to be able to predict drug metabolism and to synthesise sufficient metabolite quantities for further pharmacological testing. This study evaluates the possibility of using in vitro biotransformations to solve both these challenges in the case of testosterone as a representative component for steroids. The application of cells of Pichia pastoris with expressed membrane-associated human liver cytochrome P450 enzyme (P450) 3A4 in two cycles of a preparative-scale bioreactor experiment enabled the isolation of the common metabolites 6β-hydroxytestosterone and 6β-hydroxyandrostenedione on a 100 mg scale. Side-product formation caused by enzymes intrinsic to P. pastoris was reduced. In addition more polar testosterone metabolites formed by a P450 3A4-catalysed bioconversion, than the known mono-hydroxylated ones, are reported and 6-dehydro-15β-hydroxytestosterone as well as the di-hydroxylated steroids 6β,16β-dihydroxytestosterone, 6β,17β-dihydroxy-4-androstene-3,16-dione and 6β,12β-dihydroxyandrostenedione were isolated and verified by NMR analysis. Their respective biological significance remains to be investigated. Whole-cell P450 catalysts expressed in P. pastoris qualify as a tool for the preparative-scale synthesis of human metabolites. Biotransformation processes in combination with standard chemical procedures allow the isolation and characterisation even of minor drug metabolite products. (Figure presented.).
- Fessner, Nico D.,Srdi?, Matic,Weber, Hansj?rg,Schmid, Christian,Sch?nauer, David,Schwaneberg, Ulrich,Glieder, Anton
-
p. 2725 - 2738
(2020/06/03)
-
- Oxidative Diversification of Steroids by Nature-Inspired Scanning Glycine Mutagenesis of P450BM3 (CYP102A1)
-
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.
-
p. 8334 - 8343
(2020/09/18)
-
- Biotransformation of testosterone by Cladosporium sphaerospermum
-
Incubation of testosterone 1 with Cladosporium sphaerospermum MRC 70266 afforded six metabolites and two of these metabolites, 6β,16β,17β-trihydroxyandrost-4-en-3-one 6 and 6β,12β,17β-trihydroxyandrost-4-en-3-one 7, were determined as new compounds. The fungus mainly hydroxylated testosterone 1 at C-6β, accompanied by some minor hydroxylations at C-7β, C-12β, C-15α and C-16β. A minor oxidation at C-17 and a minor 5α-reduction were also observed.
- Yildirim, Kudret,Kuru, Ali,Y?lmaz, ?engül
-
p. 409 - 413
(2019/04/10)
-
- Biotransformation of androst-4-ene-3,17-dione by some fungi
-
The incubations of androst-4-ene-3,17-dione with Aspergillus candidus MRC 200634, Aspergillus tamarii MRC 72400, Aspergillus wentii MRC 200316 and Mucor hiemalis MRC 70325 for 5 days are reported. A. candidus MRC 200634 mainly hydroxylated androst-4-ene-3,17-dione at C-11α, C-15α and C-15β whilst A. wentii MRC 200316 hydroxylated it mainly at C-6β. A. tamarii MRC 72400 showed predominately a Baeyer–Villiger monooxygenase activity. M. hiemalis MRC 70325 hydroxylated the substrate at C-14α and reduced most of it at C-17.
- Yildirim, Kudret,Kuru, Ali,Keskin, Ece,Salihoglu, Aylin,Bukum, Neslihan
-
p. 594 - 597
(2017/11/14)
-
- Drug Oxidation by Cytochrome P450BM3: Metabolite Synthesis and Discovering New P450 Reaction Types
-
There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.
- Ren, Xinkun,Yorke, Jake A.,Taylor, Emily,Zhang, Ting,Zhou, Weihong,Wong, Luet Lok
-
p. 15039 - 15047
(2015/10/20)
-
- Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution
-
A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C - H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C - H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96 - 97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.
- Kille, Sabrina,Zilly, Felipe E.,Acevedo, Juan P.,Reetz, Manfred T.
-
scheme or table
p. 738 - 743
(2012/02/15)
-
- Structure and stereochemistry of products of hydroxylation of human steroid hormones by a housefly cytochrome P450 (CYP6A1)
-
The structure and stereochemistry of nine steroid metabolites isolated in quantities ranging from 0.15 to 1.8 mg were determined using a variety of NMR techniques, including heteronunclear multiple bond correlation (HMBC) using broadband adiabetic 13C pulses and phase-sensitive data presentation. Testosterone, and rostenedione and progesterone were oxidized with housefly cytochrome P450 6A1 enzyme reconstituted in vitro with housefly NADPH cytochrome P450 reductase and cytochrome b5. NMR analysis in CD3OD using a modified HMBC sequence as well as 2D heteronuclear single quantum correlation (HSQC), COSY and nuclear Overhauser and exchange spectroscopy (NOESY), combined with a detailed analysis of J couplings showed that hydroxylation occurs exclusively on the β-face of the steroids, at positions 2, 12, and 15. Copyright
- Jacobsen, Neil E.,Koever, Katalin E.,Murataliev, Marat B.,Feyereisen, Rene,Walker, F. Ann
-
p. 467 - 474
(2007/10/03)
-
- Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system
-
Two hundred thirteen cytochrome P450 (P450) genes were collected from bacteria and expressed based on an Escherichia coli expression system to test their hydroxylation ability to testosterone. Twenty-four P450s stereoselectively monohydroxylated testosterone at the 2α-, 2β-, 6β-, 7β-, 11β-, 12β-, 15β-, 16β-, and 17-positions (17-hydroxylation yields 17-ketoproduct). The hydroxylation site usage of the P450s is not the same as that of human P450s, while the 2α-, 2β-, 6β-, 11β-, 15β-, 16α-, and 17-hydroxylation are reactions common to both human and bacterial P450s. Most of the testosterone hydroxylation catalyzed by bacterial P450s is on the β face.
- Agematu, Hitosi,Matsumoto, Naoki,Fujii, Yoshikazu,Kabumoto, Hiroki,Doi, Satoru,Machida, Kazuhiro,Ishikawa, Jun,Arisawa, Akira
-
p. 307 - 311
(2008/02/10)
-
- Steroid transformations with Fusarium oxysporum var. cubense and Colletotrichum musae
-
The utility of two locally isolated fungi, pathogenic to banana, for steroid biotransformation has been studied. The deuteromycetes Fusarium oxysporum var. cubense (IMI 326069, UAMH 9013) and Colletotrichum musae (IMI 374528, UAMH 8929) had not been examined previously for this potential. In general, F. oxysporum var. cubense effected 7α hydroxylation on 3β-hydroxy- Δ5-steroids, 6β, 12β, and 15α hydroxylation on steroidal-4-ene-3-ones, side-chain degradation on 17α,21-dihydroxypregnene-3,20-diones, and 15α hydroxylation on estrone. Both strains were shown to perform redox reactions on alcohols and ketones.
- Wilson, Maureen R.,Gallimore, Winklet A.,Reese, Paul B.
-
p. 834 - 843
(2007/10/03)
-
- Chemical synthesis of 15β-hydroxytestosterone and its derivatives using a (4-methoxyphenyl)methyl protecting group
-
Reaction of 3β-hydroxyandrosta-5,15-dien-17-one with 4-methoxybenzyl alcohol followed by acetylation gave mainly 15β-[(4-methoxyphenyl)methoxy]- 17-oxoandrost-5-en-3β-yl acetate. This product was transformed by borohydrate reduction and organosilyl derivatization into the orthogonally protected 17β-(dimethylthexylsiloxy)-15β-[(4-methoxyphenyl)methoxy]androst- 5-en-3β-yl acetate and 17β-(dimethylisopropylsiloxy)-15β-[4- methoxyphenyl]methoxylandrost-5-en-3β-yl acetate and 17β- (dimethylisopropylsiloxy)-15β-[4-methoxyphenyl)methoxy]androst-5-en-3β-yl acetate. After deacetylation, these intermediates were submitted to Oppenauer oxidation and both yielded testosterone derivatives 17β- (dimethylthexylsiloxy)-15β-[(4-methoxyphenyl)methoxy]androst-4-en-one and 17β-(dimethylisopropylsiloxy)-15β-[(4-methoxyphenyl)-methoxy]androst-4-en- 3-one. Removal of the (4-methoxyphenyl)methyl group from position 15 by 2.3- dichloro-5,6-dicyano-1,4-benzoquinone treatment gave the partially protected derivatives 17β-(dimethylthexylsiloxy)-15β-hydroxyandrost-4-en-3-one and 17β-(dimethylisopropylsiloxy)-15β-hydroxyandrost-4-en-3-one. After acidic deprotection, the dimethylthexylsilyl derivative yielded 15β- hydroxystestosterone (15β,17β-dihydroxyandrost-4-en-3-one). Dimethylisopropylsilyl derivative was converted to the corresponding 15- hemisuccinate and 15-hemiglutarate (17β-hydroxy-3-oxoandrost-4-en-15β-yl 15-hemisuccinate and 15-hemiglutarate, respectively), which were designed as model haptens for immunoassays studies.
- Cerny, Ivan,Fajkos, Jan,Pouzar, Vladimir
-
-