3CV9

Crystal structure of vitamin D hydroxylase cytochrome P450 105A1 (R73A/R84A mutant) in complex with 1alpha,25-dihydroxyvitamin D3

Summary for 3CV9

• Entry DOI: 10.2210/pdb3cv9/pdb
• Related: 2ZBX 2ZBY 2ZBZ 3CV8
• Descriptor: Cytochrome P450-SU1, PROTOPORPHYRIN IX CONTAINING FE, 5-{2-[1-(5-HYDROXY-1,5-DIMETHYL-HEXYL)-7A-METHYL-OCTAHYDRO-INDEN-4-YLIDENE]-ETHYLIDENE}-4-METHYLENE-CYCLOHEXANE-1,3-DIOL, ... (4 entities in total)
• Functional Keywords: p450, beta prism, heme, iron, metal-binding, monooxygenase, oxidoreductase
• Biological source: Streptomyces griseolus
• Total number of polymer chains: 1
• Total formula weight: 45938.75

Authors

Hayashi, K.,Sugimoto, H.,Shinkyo, R.,Yamada, M.,Ikeda, S.,Ikushiro, S.,Kamakura, M.,Shiro, Y.,Sakaki, T. (deposition date: 2008-04-18, release date: 2008-11-04, Last modification date: 2023-11-01)

Primary citation

Hayashi, K.,Sugimoto, H.,Shinkyo, R.,Yamada, M.,Ikeda, S.,Ikushiro, S.,Kamakura, M.,Shiro, Y.,Sakaki, T.
Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1
Biochemistry, 47:11964-11972, 2008

PubMed Abstract: CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.

PubMed: 18937506
DOI: 10.1021/bi801222d

Experimental method

X-RAY DIFFRACTION (1.7 Å)