کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8337875 | 1540970 | 2018 | 36 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Properties of purified CYP2R1 in a reconstituted membrane environment and its 25-hydroxylation of 20-hydroxyvitamin D3
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کلمات کلیدی
DOPCdimyristoyl phosphatidylcholineCYP2R1HP-β-CDRORαRORγ2-hydroxypropyl-β-cyclodextrindMPCPORCyP1α,25-dihydroxyvitamin D3 - 1α، 25-دی هیدروکسوییتامین D31,25(OH)2D3 - 1،25 (OH) 2D325(OH)D3 - 25 (OH) D325-hydroxyvitamin D3 - 25 هیدروکسی ویتامین D325-hydroxylase - 25 هیدروکسیلازDioleoyl phosphatidylcholine - دیویلیل فسفاتیدیل کولینDioleoyl phosphatidylethanolamine - دیویلیل فسفاتیدیلتانولامینCytochrome P450 - سیتوکروم پی۴۵۰Phospholipid - فسفولیپیدPhospholipid vesicles - فسفولیپید مایعVitamin D3 - ویتامین D3، کوله کلسیفرولDOPE - پیش بینی کردن
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower kcat than CYP27A1 but a much lower Km for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (kcat/Km), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH)2D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH)2D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 20,25(OH)2D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: The Journal of Steroid Biochemistry and Molecular Biology - Volume 177, March 2018, Pages 59-69
Journal: The Journal of Steroid Biochemistry and Molecular Biology - Volume 177, March 2018, Pages 59-69
نویسندگان
Chloe Y.S. Cheng, Tae-Kang Kim, Saowanee Jeayeng, Andrzej T. Slominski, Robert C. Tuckey,