CYP105A1二重変異体を発現する放線菌を用いた活性型ビタミンD_3の生産

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Our previous studies revealed that the double mutant of CYP105A1 from Streptomyces griseolus, R73V/R84A, has a high ability to convert vitamin D_3(VD_3) to its biologically active form, 1α,25-dihydroxyvitamin D_3(1α,25(OH)_2D_3), suggesting the possibility of practical application of R73V/R84A to produce 1α,25(OH)_2D_3. Thus, we examined the expression of R73V/R84A in Streptomyces lividans TK23. As expected, the metabolites 25(OH)D_3 and 1α,25(OH)_2D_3 were detected in the recombinant S. lividans cell culture. In addition, novel polar metabolites 1α,25(R),26(OH)_3D_3 and 1α,25(S),26(OH)_3D_3, both of which were known to have a high antiproliferative activity and a low calcemic activity, were observed at a ratio of 5:1. The crystal structure of the double mutant with 1α,25(OH)_2D_3 and a conformational search of 1α,25(OH)_2D_3 in its active site strongly suggest that a novel hydrogen-bond network including 1α-hydroxyl group and several water molecules plays an important role in the substrate-binding for 26-hydroxylation. In conclusion, we demonstrated that R73V/R84A can catalyze hydroxylations at C25-, C1- and C26 (27) positions of VD_3 to produce biologically useful compounds.
2011-03-25