Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES.
The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1alpha,25(OH)D3. Both 1alpha-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1alpha(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1alpha(OH)D3 were estimated to be 1.7 microM and 0.51 mol/min/mol P450, respectively, while those for 1alpha-hydroxylation activity toward 25(OH)D3 were 0.050 microM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1alpha-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1alpha,25(OH)D3, 1alpha-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.
Uchida
Eriko
E
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan.
Kagawa
Norio
N
Sakaki
Toshiyuki
T
Urushino
Naoko
N
Sawada
Natsumi
N
Kamakura
Masaki
M
Ohta
Miho
M
Kato
Shigeaki
S
Inouye
Kuniyo
K
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D018835:Chaperonin 10 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D018834:Chaperonin 60 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D018833:Chaperonins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli; D024861:Genetic Enhancement; D051379:Mice; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity
D018835:Chaperonin 10; D018834:Chaperonin 60; D011994:Recombinant Proteins; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D018833:Chaperonins