title: Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase.
abstract: Vitamin D(3) hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D(3) (VD(3)) to 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)VD(3)) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD(3), i.e. the conversion of VD(3) to 25-hydroxyvitamin D(3) (25(OH)VD(3)) and then of 25(OH)VD(3) to 1α,25(OH)(2)VD(3), a hormonal form of VD(3). Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD(3)-bound, and 25(OH)VD(3)-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substrate-bound structure of Vdh-K1 accommodates both VD(3) and 25(OH)VD(3) but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD(3) hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD(3) 25-hydroxylase and 25(OH)VD(3) 1α-hydroxylase activities.
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Sapporo 062-8517, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Yasutake
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Fujii
authors.forename: Taiki
authors.initials: T
authors.lastname: Nishioka
authors.forename: Woo-Kwang
authors.initials: WK
authors.lastname: Cheon
authors.forename: Akira
authors.initials: A
authors.lastname: Arisawa
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tamura
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002117:Calcitriol / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002762:Cholecalciferol / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002762:Cholecalciferol; D013250:Steroid Hydroxylases; D002117:Calcitriol