title: 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 D(3) to its biologically active form, 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)], suggesting the possibility for R73V/R84A to produce 1α,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 1α,25(OH)(2)D(3) were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,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 1α,25(R),26(OH)(3)D(3) and 1α,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 1α,25(OH)(2)D(3) and a docking model of 1α,25(OH)(2)D(3) 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 D(3) to produce biologically useful compounds.
authors: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Kurokawa, Imizu, Toyama, Japan.
authors: Keiko
authors: K
authors: Hayashi
authors: Kaori
authors: K
authors: Yasuda
authors: Hiroshi
authors: H
authors: Sugimoto
authors: Shinichi
authors: S
authors: Ikushiro
authors: Masaki
authors: M
authors: Kamakura
authors: Atsushi
authors: A
authors: Kittaka
authors: Ronald L
authors: RL
authors: Horst
authors: Tai C
authors: TC
authors: Chen
authors: Miho
authors: M
authors: Ohta
authors: Yoshitsugu
authors: Y
authors: Shiro
authors: Toshiyuki
authors: T
authors: Sakaki
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D002762:Cholecalciferol / Q000378:metabolism*; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005818:Genetic Engineering; D014644:Genetic Variation; D006900:Hydroxylation; D007700:Kinetics; D013058:Mass Spectrometry; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D011994:Recombinant Proteins / Q000378:metabolism; D048170:Streptomyces lividans / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; C086254:P450SU1 protein, Streptomyces griseolus