Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1: Enzymes and catalysis

Abstract

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D3 to its biologically active form, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], suggesting the possibility for R73V/R84A to produce 1α,25(OH)2D3. 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 D3 [25(OH)D3] and 1α,25(OH) 2D3 were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)2D3, the second-step metabolite of vitamin D3, was observed, although a considerable amount of vitamin D3 still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)3D3 and 1α,25(S),26(OH)3D3, 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 1α,25(OH)2D3 and a docking model of 1α,25(OH)2D3 in its active site strongly suggest a hydrogen-bond network including the 1α-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 D3 to produce biologically useful compounds. © 2010 FEBS.