Chloe Y.S. ChengTae Kang KimSaowanee JeayengAndrzej T. SlominskiRobert C. TuckeyBirmingham VA Medical CenterUniversity of Western AustraliaThe University of Alabama at BirminghamFaculty of Medicine, Siriraj Hospital, Mahidol University2019-08-232019-08-232018-03-01Journal of Steroid Biochemistry and Molecular Biology. Vol.177, (2018), 59-6918791220096007602-s2.0-85027251396https://repository.li.mahidol.ac.th/handle/20.500.14594/45231© 2017 Elsevier Ltd 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 k cat than CYP27A1 but a much lower K m for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (k cat /K m ), 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) 2 D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH) 2 D3 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) 2 D3]. The 20,25(OH) 2 D3 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1016/j.jsbmb.2017.07.011