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Production of an active form of vitamin D 2 by genetically engineered CYP105A1.

Authors :: Yasuda K
Yogo Y
Sugimoto H
Mano H
Takita T
Ohta M
Kamakura M
Ikushiro S
Yasukawa K
Shiro Y
Sakaki T
Source :: Biochemical and biophysical research communications [Biochem Biophys Res Commun] 2017 Apr 29; Vol. 486 (2), pp. 336-341. Date of Electronic Publication: 2017 Mar 14.
Publication Year :: 2017
Abstract: Our previous studies revealed that CYP105A1 can convert vitamin D <subscript>3</subscript> (VD3) to its active form, 1α,25-dihydroxyvitamin D <subscript>3</subscript> (1,25D3). Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity; the activity of double variants R73A/R84A and R73A/R84V was more than 100-fold higher than that of the wild type of CYP105A1. In contrast, these variants had a low ability to convert vitamin D <subscript>2</subscript> (VD2) to 1α,25-dihydroxyvitamin D <subscript>2</subscript> (1,25D2), whereas they catalyzed the sequential hydroxylation at positions C25 and C26 to produce 25,26D2. A comparison of the docking models of 25D2 and 25D3 into the substrate-binding pocket of R73A/R84A suggests that the side chain of the Met239 inhibits the binding of 25D2 for 1α-hydroxylation. Therefore, the Met239 residue of R73A/R84A was substituted for Ala. As expected, the triple variant R73A/R84A/M239A showed a 22-fold higher 1α-hydroxylation activity towards 25D2. To the best of our knowledge, this is the first report on the generation of microbial cytochrome P450 that converts VD2 to 1,25D2 via 25D2.<br /> (Copyright © 2017 Elsevier Inc. All rights reserved.)