Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1
Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1
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DOI：
10.1111/j.1742-4658.2010.07791.x
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发表时间：
2010-10-01
期刊：
影响因子：
5.4
通讯作者：
Sakaki, Toshiyuki
中科院分区：
文献类型：
--
作者：
Hayashi, Keiko;Yasuda, Kaori;Sakaki, Toshiyuki
Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D-3 to its biologically active form, 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(OH)(2)D-3], suggesting the possibility for R73V/R84A to produce 1 alpha,25(OH)(2)D-3. Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25-hydroxyvitamin D-3 [25(OH)D-3] and 1 alpha,25(OH)(2)D-3 were detected in the cell culture of the recombinant S. lividans. A large amount of 1 alpha,25(OH)(2)D-3, the second-step metabolite of vitamin D-3, was observed, although a considerable amount of vitamin D-3 still remained in the culture. In addition, novel polar metabolites 1 alpha,25(R),26(OH)(3)D-3 and 1 alpha,25(S),26(OH)(3)D-3, both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5 : 1. The crystal structure of the double variant with 1 alpha,25(OH)(2)D-3 and a docking model of 1 alpha,25(OH)(2)D-3 in its active site strongly suggest a hydrogen-bond network including the 1 alpha-hydroxyl group, and several water molecules play an important role in the substrate-binding for 26-hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D-3 to produce biologically useful compounds.
