- Microbial transformation of androstenedione by Cladosporium sphaerospermum and Ulocladium chartarum
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In this work, incubations of androstenedione 1 with Cladosporium sphaerospermum MRC 70266 and Ulocladium chartarum MRC 72584 have been reported. C. sphaerospermum MRC 70266 mainly hydroxylated 1 at C-6β, accompanied by a hydroxylation at C-15α, a reduction at C-17, a 5α-reduction and oxidations at C-6 and C-16 following hydroxylations. U. chartarum MRC 72584 hydroxylated 1 at C-6β, C-7α, C-7β and C-14α, accompanied by an oxidation at C-6 following its hydroxylation, a reduction at C-17 and a 5α-reduction. 6β,17β-Dihydroxyandrost-4-en-3,16-dione 8, one of the metabolites from the incubation of 1 with C. sphaerospermum MRC 70266, was determined as a new compound.
- Yildirim, Kudret,Kuru, Ali,Kü?ükba?ol, Eda
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- Cytochrome P450 metabolic activities in the small intestine of cynomolgus macaques bred in cambodia, china, and indonesia
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Summary: Cynomolgus macaques, used in drug metabolism studies due to their evolutionary closeness to humans, are mainly bred in Asian countries, including Cambodia, China, and Indonesia. Cytochromes P450 (P450s) are important drug-metabolizing enzymes, present in the liver and small intestine, major drug metabolizing organs. Previously, our investigation did not find statistically significant differences in hepatic P450 metabolic activities measured in cynomolgus macaques bred in Cambodia (MacfaCAM) and China (MacfaCHN). In the present study, P450 metabolic activity was investigated in the small intestine of MacfaCAM and MacfaCHN, and cynomolgus macaques bred in Indonesia (MacfaIDN) using P450 substrates, including 7-ethoxyresorufin, coumarin, bupropion, paclitaxel, diclofenac, S-mephenytoin, bufuralol, chlorzoxazone, and testosterone. The results indicated that P450 metabolic activity of the small intestine was not statistically significantly different (2.0-fold) in MacfaCAM, MacfaCHN, and MacfaIDN. In addition, statistically significant sex differences were not observed (2.0-fold) in any P450 metabolic activity in MacfaCAM as supported by mRNA expression results. These results suggest that P450 metabolic activity of the small intestine does not significantly differ statistically among MacfaCAM, MacfaCHN, and MacfaIDN.
- Nakanishi, Yasuharu,Yamashita, Hiroyuki,Yoshikawa, Tsuyoshi,Tominaga, Takeshi,Nojiri, Koichiro,Sunaga, Yoshiharu,Muneoka, Atsunobu,Iwasaki, Kazuhide,Utoh, Masahiro,Nakamura, Chika,Yamazaki, Hiroshi,Uno, Yasuhiro
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- Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation
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The human cytochrome P450 2D6 (CYP2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone - just as other steroids - is an atypical substrate and only poorly metabolized by CYP2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in Pichia pastoris. Employing whole-cell biotransformations coupled with HPLC-MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP2D6 variant was determined. Covering the whole sequence space, CYP2D6 variants with improved activity and so far unknown regio-preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2β-position, while the slow formation of 6β-hydroxytestosterone, the main product of wild-type CYP2D6, was further reduced. Two point mutations have already been sufficient to convert CYP2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild-type CYP2D6. Furthermore, this study is also an example for efficient human CYP engineering in P. pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism. 400 cytochrome P450 2D6 (CYP2D6) variants representing all possible amino acid exchanges at two important enzyme's residues were expressed and individually analyzed to investigate their influence on regioselective steroid hydroxylation. Steroids represent a substrate class atypical for wildtype CYP2D6. Employing this strategy CYP2D6 variants with improved activity and variants with altered region-preference were identified and characterized.
- Geier, Martina,Braun, Andreas,Fladischer, Patrik,Stepniak, Piotr,Rudroff, Florian,Hametner, Christian,Mihovilovic, Marko D.,Glieder, Anton
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- Influence of P450 3A4 SRS-2 residues on cooperativity and/or regioselectivity of aflatoxin B1 oxidation
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The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al. (1995) Arch. Biochem. Biophys. 317, 74-384]. P450 3A4 prefers AFB1 3α-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation. P450 3A5, on the other hand, is a relatively sluggish 3α-hydroxylase and converts AFB1 predominantly to the genotoxic exo-8,9-epoxide. Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB1 regioselectivity toward that of P450 3A5 [Wang et al. (1998) Biochemistry 37, 12536-12545]. In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB1 detoxification via 3α-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB1 oxidation. Molecular dynamics analyses with an existing P450 3A4 homology model [Szklarz and Halpert (1997) J. Comput. Aided Mol. Des. 11,265] suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity. To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB1 3α-hydroxylation. However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses. Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB1 molecular modeling analyses with a newly refined P450 3A4 homology model. These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB1 binding at the active site in either orientation leading to 3α- or exo-8,9-oxidation. Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB1 3α-hydroxylation and exo-8,9-oxidation.
- Xue, Linlong,Wang, Huifen Faye,Wang, Qinmi,Szklarz, Grazyna D.,Domanski, Tammy L.,Halpert, James R.,Correia, Maria Almira
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- Biotransformations of steroid compounds by Chaetomium sp. KCH 6651
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Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product-6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.
- Janeczko, Tomasz,Dmochowska-Gladysz, Jadwiga,Kostrzewa-Suslow, Edyta,Bialonska, Agata,Ciunik, Zbigniew
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- Microbial transformation of steroids: Contribution to 14α-hydroxylations
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The regioselective and stereoselective hydroxylation of steroids by fungal strains previously known for their hydroxylation capabilities, such as Thamnostylum (= Helicostylum) piriforme ATCC 8992, Mucor griseocyanus ATCC 1207a, Actinomucor elegans (= Mucor parasiticus) MMP 3122 (Mucorales), and Zygodesmus sp. ATCC 14716, was investigated with special interest for the 14α-hydroxylation reaction. A preliminary screening had shown that some of these microorganisms were adequate for the production of 14α-hydroxylated derivatives of the following steroids: progesterone, 5β-pregnane-3,20- dione, 3β-hydroxy-5β-pregnane-20-one, 3β-hydroxy-5β-17(αH)-etianic acid methyl ester, androst-4-ene-3,17-dione, and testosterone. About 20 metabolites have been isolated and purified by silicagel chromatography and semi-preparative reverse-phase HPLC. These metabolites have been fully characterized by 1H, 13C NMR and mass spectrometry. All the identified metabolites were hydroxylated at some distinct positions, such as 6β-, 7α- , 9α-, 14α-, 15β-, or dihydroxylated at 6β, 14α-, 7α, 14α-, 9α, 14α- , 14α, 15α-, 14α, 15β-positions; nine of these metabolites have not been reported previously. The relationship between the structural features of the investigated steroids and the site-specific hydroxylation has been delineated, and progesterone was found to be the best substrate for the production of 14α-hydroxylated derivative, using T. piriforme.
- Azerad, Robert
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- Preparative-Scale Production of Testosterone Metabolites by Human Liver Cytochrome P450 Enzyme 3A4
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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
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p. 2725 - 2738
(2020/06/03)
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- Biotransformation of testosterone by Cladosporium sphaerospermum
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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
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p. 409 - 413
(2019/04/10)
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- Microbial transformation of epiandrosterone by Aspergillus sydowii
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Incubation of epiandrosterone with Aspergillus sydowii MRC 200653 afforded ten metabolites. The fungal dehydrogenation of epiandrosterone is reported for the first time. The formation of the major metabolite, 6?-hydroxyandrost-4-ene-3,17-dione, involved first dehydrogenation to give a 4-ene and then hydroxylation at C-6?. Small amounts of the substrate were hydroxylated at C-1α, C-7α, C-7β and C-11α.
- Yildirim, Kudret,Kuru, Ali
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p. 718 - 721
(2016/12/30)
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- The generation of a steroid library using filamentous fungi immobilized in calcium alginate Dedicated to the memory of Professor Sir John W. Cornforth, University of Sussex (1917-2013).
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Four fungi, namely, Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, and Whetzelinia sclerotiorum ATCC 18687, were subjected to entrapment in calcium alginate, and the beads derived were used in the biotransformation of the steroids 3β,17β-dihydroxyandrost-5-ene (1) and 17β-hydroxyandrost-4-en-3-one (2). Incubations performed utilized beads from two different encapsulated fungi to explore their potential for the production of metabolites other than those derived from the individual fungi. The investigation showed that steroids from both single and crossover transformations were typically produced, some of which were hitherto unreported. The results indicated that this general technique can be exploited for the production of small libraries of compounds.
- Peart, Patrice C.,Reynolds, William F.,Reese, Paul B.
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- Biotransformation of testosterone and testosterone heptanoate by four filamentous fungi
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The microbial transformations of testosterone and testosterone heptanoate by four fungi: Absidia griseolla var. igachii PTCC 5260, Acremonium chrysogenu PTCC 5271, Fusarium fujikuroi PTCC 5144, and Fusarium solani complex PTCC 5285 were investigated for the first time. Incubation of testosterone heptanoate with F. fujikuroi and F. solani yielded three metabolites, which were isolated and characterized as testosterone, androst-4-ene-3,17-dione, and 6β-hydroxy testosterone. 6β-Hydroxy testosterone was the major metabolite obtained from testosterone heptanoate biotransformation by two fungal species. A. griseolla and A. chrysogenu produced 14α-hydroxy testosterone as major metabolite, together with testosterone and 6β-hydroxy testosterone in lower yields. The biotransformation of testosterone by F. fujikuroi and A. griseolla was also investigated in order to examine the influence of the ester group on the course of transformation. Androst-4-ene-3,17-dione was only identified in the biotransformation of testosterone by F. fujikuroi. The same product was observed in incubation of testosterone by A. griseolla, together with 14α-hydroxy testosterone in very low yield. Furthermore, time course study was also carried out in order to examine the formation of metabolites as a function of time, which was determined by HPLC. The structures of compounds were determined by their comprehensive spectroscopic analysis and comparison with literature data.
- Ghasemi, Sabrieh,Mohajeri, Maryam,Habibi, Zohreh
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supporting information
p. 7 - 12
(2014/12/11)
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- Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution
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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.
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p. 738 - 743
(2012/02/15)
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- Steroid transformations with Fusarium oxysporum var. cubense and Colletotrichum musae
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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.
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p. 834 - 843
(2007/10/03)
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