- Investigation of Vitamin D2 and Vitamin D3 Hydroxylation by Kutzneria albida
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The active vitamin D metabolites 25-OH?D and 1α,25-(OH)2?D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D2 (VD2) and vitamin D3 (VD3) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25-OH?D2 and 25-OH?D3 was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100-fold higher product concentrations of 25-OH?D3 and tenfold higher concentrations of 25-OH?D2 after addition of 5 % (w/v) 2-hydroxypropyl β-cyclodextrin (2-HPβCD) were reached. Besides the single-hydroxylation products, the human double-hydroxylation products 1,25-(OH)2?D2 and 1,25-(OH)2?D3 and various other potential single- and double-hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD2 and VD3 metabolites.
- Schmitz, Lisa Marie,Kinner, Alina,Althoff, Kirsten,Rosenthal, Katrin,Lütz, Stephan
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p. 2266 - 2274
(2021/04/26)
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- Highly regio- and stereoselective hydroxylation of vitamin D2 by CYP109E1
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Vitamin D2 is a form of vitamin D derived from mushrooms and plants which is structurally modified in the body due to the action of several enzymes. The resulting metabolites represent important compounds with potential bioactive properties. However, they are poorly studied and their availability is mostly limited. In order to identify new enzymes capable of producing vitamin D2 metabolites, we investigated a bacterial P450 monooxygenase, CYP109E1, which was previously shown to be a vitamin D3 hydroxylase. It was found that CYP109E1 catalyzes a vitamin D2 two-step hydroxylation at positions C24 and C25 resulting in the generation of 24(R),25-diOH VD2. Interestingly, the enzyme showed high selectivity towards vitamin D2, whereas it showed an unselective product pattern for the structurally similar vitamin D3. Our docking results for vitamin D2 and D3 revealed favorable hydroxylation positions for both substrates and suggested an explanation for the high selectivity of CYP109E1 towards vitamin D2. In addition, we established a whole-cell biocatalyst expressing CYP109E1 in Bacillus megaterium to produce 24(R),25-diOH VD2 and a production yield of 12.3 ± 1.2 mg/L was obtained after 48 h. To the best of our knowledge, this is the first report on the generation of 24(R),25-diOH VD2 by a microbial biocatalyst allowing a low-cost and eco-friendly production of this pharmaceutically interesting and expensive metabolite from the relatively cheap substrate, VD2.
- Putkaradze, Natalia,K?nig, Lisa,Kattner, Lars,Hutter, Michael C.,Bernhardt, Rita
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p. 295 - 300
(2020/01/31)
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- Molecular determinants for selective C25-hydroxylation of vitamins D2 and D3 by fungal peroxygenases
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Hydroxylation of vitamin D by Agrocybe aegerita and Coprinopsis cinerea peroxygenases was investigated in a combined experimental and computational study. 25-Monohydroxylated vitamins D3 (cholecalciferol) and D2 (ergocalciferol), compounds of high interest in human health and animal feeding, can be obtained through a reaction with both fungal enzymes. Differences in conversion rates, and especially in site selectivity, were observed. To rationalize the results, diffusion of D2 and D3 on the molecular structure of the two enzymes was performed using the PELE software. In good agreement with experimental conversion yields, simulations indicate more favorable energy profiles for the substrates' entrance in C. cinerea than for A. aegerita enzyme. On the other hand, GC-MS analyses show that while a full regioselective conversion of D2 and D3 into the active C25 form is catalyzed by C. cinerea peroxygenase, A. aegerita yielded a mixture of the hydroxylated D3 products. From the molecular simulations, relative distance distributions between the haem compound I oxygen atom and H24/H25 atoms (hydrogens on C24 and C25, respectively) were plotted. Results show large populations for O-H25 distances below 3 ? for D2 and D3 in C. cinerea in accordance with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 ?) for O-H25 and O-H24, which can justify the hydroxylation observed in C24. In the case of ergocalciferol, due to the bulky methyl group in position C24, very few structures are found with O-H24 distances below 3 ? and thus, as expected, the reaction was only observed at the C25 position.
- Lucas, Fátima,Babot, Esteban D.,Ca?ellas, Marina,Del Río, José C.,Kalum, Lisbeth,Ullrich, René,Hofrichter, Martin,Guallar, Victor,Martínez, Angel T.,Gutiérrez, Ana
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p. 288 - 295
(2015/12/31)
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- Regioselective hydroxylation in the production of 25-hydroxyvitamin D by Coprinopsis cinerea peroxygenase
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Monohydroxylated metabolites of vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol), generically known as 25-hydroxycalciferol, are better for several diseases, and other applications, than vitamin D (calciferol). This work describes a novel biotechnological approach for the preparation of 25-hydroxycalciferols, starting from readily available cholecalciferol and ergocalciferol. This approach enables the regioselective (100%) hydroxylation of these compounds (at the C-25 position) under mild and environmentally friendly conditions by using a peroxidase from the fungus Coprinopsis cinerea (gene model CC1G-08427T0 from the sequenced genome), which catalyzes monooxygenation with H2O2 as the only co-substrate (peroxygenase). Hydroxylation of cholecalciferol and ergocalciferol is a true peroxygenation, as demonstrated by incorporation of 18O from H218O2 into the products. The peroxygenase has additional advantages related to its recombinant nature, enabling enzyme engineering and low-cost overexpression in an industrial host. Therefore, the peroxygenase is a promising biocatalyst for the production of vitamin D active metabolites.
- Babot, Esteban D.,Del R??o, Jos?? C.,Kalum, Lisbeth,Mart??nez, Angel T.,Guti??rrez, Ana
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p. 283 - 290
(2015/02/19)
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- Characterization of rat and human CYP2J enzymes as Vitamin D 25-hydroxylases
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vitamin D is 25-hydroxylated in the liver, before being activated by 1α-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848-56]. In this study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087 min-1), vitamin D2 (0.16 min-1), and 1α-hydroxyvitamin D3 (2.2 min-1). Interestingly, human CYP2J2 hydroxylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4 min-1) more efficiently than vitamin D2 (0.86 min-1). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3 and D2.
- Aiba, Isamu,Yamasaki, Tomoaki,Shinki, Toshimasa,Izumi, Shunsuke,Yamamoto, Keiko,Yamada, Sachiko,Terato, Hiroaki,Ide, Hiroshi,Ohyama, Yoshihiko
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p. 849 - 856
(2007/10/03)
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- Immunosuppressive agents
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The invention relates to pharmaceutical compositions comprising at least one Vitamin D derivative and a method of using the pharmaceutical compositions in suppressing immune responses.
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- Studies on the Synthesis of Side-Chain Hydroxylated Metabolites of Vitamin D. 2. Stereocontrolled Synthesis of 25-Hydroxyvitamin D2
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An efficient synthesis of 25-hydroxyvitamin D2 is described.The chiral center at C-24 was introduced by the stereospecific and regioselective displacement of an allylic carbamate by a cuprate.The triene system was assembled by Horner-Wittig coupling of ke
- Sardina, F. Javier,Mourino, Antonio,Castedo, Luis
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p. 1264 - 1269
(2007/10/02)
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- FACILE AND STEREOSELECTIVE SYNTHESIS OF 25-HYDROXYVITAMIN D2
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25-Hydroxyvitamin D2 was synthesized conveniently and stereoselectively for the first time by utilizing Sharpless' chiral epoxidation and the subsequent regio- and stereoselective methylation with lithium dimethylcuprate to introduce the desire
- Yamada, Sachiko,Shiraishi, Masami,Ohmori, Masayuki,Takayama, Hiroaki
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p. 3347 - 3350
(2007/10/02)
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