19356-17-3Relevant articles and documents
Optimization of bioconversion conditions for vitamin D3 to 25-hydroxyvitamin D using Pseudonocardia autotrophica CGMCC5098
Luo, Jinqi,Jiang, Fang,Fang, Weizhen,Lu, Qun
, p. 11 - 18 (2017)
The optimal culture conditions for bioconversion of vitamin D3 to calcifediol (25(OH)D3) were investigated by varying carbon and nitrogen sources, metal salt concentrations, initial pH, temperature, solvents, surfactants, and agitation speed. In the process of this microbial hydroxylation, the timing of the addition of vitamin D3, which is dissolved in ethanol, is of critical importance. Besides, the concentration of ethanol in zymotic fluid is the key factor to get high conversion ratio of vitamin D3. In particular, the optimal culture conditions were 1.5% glucose, 1.5% soybean cake meal, 0.5% yeast extract, 0.5% corn steep liquor, 0.3% CaCO3, 0.1% NaCl, 0.2% KH2PO4, pH 7.2 at 27 °C and the timing of the addition of vitamin D3 dissolved in 5% (v/v) ethanol was 48 h followed by the inoculation of seed culture broth. Under the optimized conditions, the conversion of vitamin D3 (1 g/L) by Pseudonocardia autotrophica CGMCC5098 in 50 L fermenter resulted in about 61.31% bioconversion ratio (639 mg/L) of 25(OH)D3 on the 5th day.
Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes
Fujii, Tadashi,Fujii, Yoshikazu,Machida, Kazuhiro,Ochiai, Atsushi,Ito, Masashi
, p. 805 - 810 (2009)
We report here some efficient biotransformations using Escherichia coli strains with disruptions for the AcrAB-TolC efflux pump system. Biotransformations of compactin into pravastatin (6α-hydroxy-iso- compactin) were performed using E. coli strains with tolC and/or acrAB mutations expressing a cytochrome P450 (P450) gene. The production levels of pravastatin using strains with acrAB, tolC, and tolC acrAB mutations increased by 3.7-, 7.0-, and 7.1-fold, respectively. Likewise, the production levels of 25-hydroxy vitamin D3 and 25-hydroxy 4-cholesten 3-one using tolC acrAB mutant strains expressing an individual P450 gene increased by 2.2- and 16-fold, respectively. The enhancement of this biotransformation efficiency could be explained by increases in the intracellular amounts of substrates and the concentrations of active P450s. These results demonstrate that we have achieved versatile methods for efficient biotransformations using E. coli strains with tolC acrAB mutations expressing P450 genes.
Isolation and identification of 2α,25-dihydroxyvitamin D3, a new metabolite from Pseudonocardia autotrophica 100U-19 cells incubated with Vitamin D3
Takeda, Koji,Kominato, Kaichiro,Sugita, Atsuko,Iwasaki, Yukiko,Shimazaki, Mika,Shimizu, Masato
, p. 736 - 744 (2006)
Pseudonocardia autotrophica converted Vitamin D3 to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. The hydroxylation of Vitamin D3 with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D3 metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D3, and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2α,25-dihydroxyvitamin D3 by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments.
Investigation of Vitamin D2 and Vitamin D3 Hydroxylation by Kutzneria albida
Schmitz, Lisa Marie,Kinner, Alina,Althoff, Kirsten,Rosenthal, Katrin,Lütz, Stephan
, p. 2266 - 2274 (2021)
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.
25-HydroxyvitaminD3 Synthesis by Enzymatic Steroid Side-Chain Hydroxylation with Water
Warnke, Markus,Jung, Tobias,Dermer, Juri,Hipp, Karin,Jehmlich, Nico,Von Bergen, Martin,Ferlaino, Sascha,Fries, Alexander,Müller, Michael,Boll, Matthias
, p. 1881 - 1884 (2016)
The hydroxylation of vitaminD3 (VD3, cholecalciferol) side chains to give 25-hydroxyvitaminD3 (25OHVD3) is a crucial reaction in the formation of the circulating and biologically active forms of VD3. It is usually catalyzed by cytochrome P450 monooxygenases that depend on complex electron donor systems. Cell-free extracts and a purified Mo enzyme from a bacterium anaerobically grown with cholesterol were employed for the regioselective, ferricyanide-dependent hydroxylation of VD3 and proVD3 (7-dehydrocholesterol) into the corresponding tertiary alcohols with greater than 99 % yield. Hydroxylation of VD3 strictly depends on a cyclodextrin-assisted isomerization of VD3 into preVD3, the actual enzymatic substrate. This facile and robust method developed for 25OHVD3 synthesis is a novel example for the concept of substrate-engineered catalysis and offers an attractive alternative to chemical or O2 /electron-donor-dependent enzymatic procedures.
A single mutation at the ferredoxin binding site of P450 Vdh enables efficient biocatalytic production of 25-hydroxyvitamin D3
Yasutake, Yoshiaki,Nishioka, Taiki,Imoto, Noriko,Tamura, Tomohiro
, p. 2284 - 2291 (2013)
Vitamin D3 hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two-step hydroxylation of vitamin D3 (VD3) to produce 25-hydroxyvitamin D 3 (25(OH)VD3) and 1α,25-dihydroxyvitamin D 3 (1α,25(OH)2VD3). These hydroxylated forms of VD3 are useful as pharmaceuticals for the treatment of conditions associated with VD3 deficiency and VD3 metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin-binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD3, a promising intermediate for the synthesis of various hydroxylated VD3 derivatives, by using nisin-treated Rhodococcus erythropolis cells containing VdhT107A. The gene-expression cassette encoding Bacillus megaterium glucose dehydrogenase-IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL-1 25(OH)VD3 was successfully produced by a 2 h bioconversion. Copyright
Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes
Barckhausen, Ian R.,Child, Stella A.,Glass, Sarah M.,Goldfarb, Margo H.,Guengerich, F. Peter,Reddish, Michael J.
, (2020)
Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6–200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 μM Adx, and higher concentrations were not inhibitory up to at least 50 μM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.
Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D 3 hydroxylase
Yasutake, Yoshiaki,Fujii, Yoshikazu,Nishioka, Taiki,Cheon, Woo-Kwang,Arisawa, Akira,Tamura, Tomohiro
, p. 31193 - 31201 (2010)
Vitamin D3 hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D3 (VD3) to 1α,25-dihydroxyvitamin D3 (1α,25(OH)2VD 3) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD3, i.e. the conversion of VD3 to 25-hydroxyvitamin D3 (25(OH)VD3) and then of 25(OH)VD3 to 1α,25(OH) 2VD3, a hormonal form of VD3. Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD3-bound, and 25(OH)VD3-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substratebound structure of Vdh-K1 accommodates both VD3 and 25(OH)VD3 but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD3 hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD3 25-hydroxylase and 25(OH)VD3 1α-hydroxylase activities.
Characterization of rat and human CYP2J enzymes as Vitamin D 25-hydroxylases
Aiba, Isamu,Yamasaki, Tomoaki,Shinki, Toshimasa,Izumi, Shunsuke,Yamamoto, Keiko,Yamada, Sachiko,Terato, Hiroaki,Ide, Hiroshi,Ohyama, Yoshihiko
, p. 849 - 856 (2006)
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.
IMPROVED, COST EFFECTIVE PROCESS FOR SYNTHESIS OF VITAMIN D3 AND ITS ANALOGUE CALCIFEDIOL FROM ERGOSTEROL
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Page/Page column 49, (2021/01/29)
Disclosed herein is an improved and efficient process for synthesis of vitamin D3 and its analogue Calcifediol from Ergosterol. Particularly, the present invention discloses the synthesis of key intermediate 3β-tert-Butyldimethylsilyloxy-22-hydroxy-23,24-bisnorchola-5,7-diene (5), and novel intermediate β-tert-Butyldimethylsilyloxy-22-iodo-23,24-bisnorchola-5,7-diene (9) by a simple and cost effective process. The industrially viable processes for preparation of said intermediate(s) results in providing provitamins with various side chains and the desired products in high yield.
