Purified Mouse CYP27B1 Can Hydroxylate 20,23-Dihydroxyvitamin D₃, Producing 1α,20,23-Trihydroxyvitamin D₃, Which Has Altered Biological Activity

Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

“…CYP27B1 activity was measured with substrates incorporated into phospholipid vesicles, a system that mimics the native environment of the cytochrome in the inner mitochondrial membrane, which we have used previously with CYP27B1 (Tang et al, 2010b (Tuckey et al, 2008a;Tang et al, 2010a), and by mass spectrometry and NMR in the case of the products from 20,24(OH) 2 D3, 20,25(OH) 2 D3, and 20,26(OH) 2 D3 (described below). For 22(OH)D3 and 20,22(OH) 2 D3, where limited substrate was available and the hydroxylation rate was low, insufficient product was generated by CYP27B1 to identify their structures by NMR, but it is presumed that they are the 1a-hydroxy derivatives.…”

“…Along with the primary products of CYP11A1 action on vitamin D3, 17,20(OH) 2 D3, 20,22(OH) 2 D3, and 20,23(OH) 2 D3, we have a unique set of derivatives all containing a 20-hydroxyl group plus one additional hydroxyl group at every position on the side chain, except C21 (note that C26 and C27 are equivalent). All of these 20-hydroxy derivatives have been shown to be biologically active (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Janjetovic et al, , 2011Li et al, 2010;Slominski et al, 2010Slominski et al, , 2011Slominski et al, , 2012aTang et al, 2010a;Tuckey et al, 2011;Kim et al, 2012;Lu et al, 2012;Wang et al, 2012). The low catalytic efficiency of CYP27B1 toward 20(OH)D3 is greatly enhanced when the second hydroxyl group is added toward the end of the side chain, as seen for 24,20(OH) 2 D3, 20,25(OH) 2 D3, and 20,26(OH) 2 D3.…”

“…Previously, we reported that purified mouse CYP27B1 can 1a-hydroxylate the two major products of CYP11A1 action on vitamin D3, 20(OH)D3 and 20,23(OH) 2 D3, and one of the major products of CYP11A1 action on vitamin D2, 20(OH)D2, with the products displaying altered biological activity compared with the parent secosteroids (Tang et al, 2010a(Tang et al, ,b, 2012Slominski et al, 2011). Recently, we successfully expressed human CYP27B1 in Escherichia coli (E. coli), extracted and partially purified the enzyme, and carried out a detailed characterization of its catalytic activity on its classic substrates .…”

“…Via binding to the vitamin D receptor, these compounds can inhibit keratinocyte, melanoma, leukemia, breast, and liver cancer cell proliferation; promote differentiation; and display antiinflammatory activity by decreasing nuclear factor-ĸB activity (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Janjetovic et al, , 2011Li et al, 2010;Slominski et al, 2010Slominski et al, , 2011Slominski et al, , 2012aTang et al, 2010a;Tuckey et al, 2011;Kim et al, 2012;Lu et al, 2012;Wang et al, 2012). Importantly, all three of these derivatives lack calcemic activity in rodents, even at relatively high doses (Slominski et al, , 2013aWang et al, 2012).…”

Production of 22-Hydroxy Metabolites of Vitamin D3by Cytochrome P450scc (CYP11A1) and Analysis of Their Biological Activities on Skin Cells

“…The two major products of vitamin D 3 metabolism by P450scc, 20(OH)D 3 and 20,23(OH) 2 D 3 , have been tested on a range of skin and other cell types, and they have many but not all of the properties of the hormonally active form of vitamin D 3 , 1,25(OH) 2 D 3 , acting as partial receptor agonists (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Slominski et al, 2010;Tang et al, 2010a). Properties in common with 1,25(OH) 2 D 3 on skin cells include inhibition of proliferation and stimulation of differentiation of epidermal keratinocytes, and inhibition of nuclear factor-B activity via the stimulation of the expression of inhibitor of nuclear factor-B ␣.…”