Enzymatic studies on the key enzymes of vitamin D metabolism; 1 alpha-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24).
The key enzymes of vitamin D3 metabolism, renal 25-hydroxyvitamin D3 1 alpha-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24) were expressed in Escherichia coli, and their enzymatic properties were revealed. As expected, mouse CYP27B1 and human CYP27B1 showed the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with the Michaelis constant, Km, value of 2.7 microM. Unexpectedly, both mouse CYP27B1 and human CYP27B1 showed greater Vmax/Km values toward 24,25-dihydroxyvitamin D3 than 25-hydroxyvitamin D3, suggesting that 24, 25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for both CYP27B1. Enzymatic studies on substrate specificity of CYP27B1 revealed that 25-hydroxyl group of vitamin D3 was essential for the 1 alpha-hydroxylase activity, and 24-hydroxyl group enhanced the activity, but, 23-hydroxyl group greatly reduced the activity. On rat CYP24, it was demonstrated that CYP24 catalyzed four-step monooxygenation towards 25-hydroxyvitamin D3. Furthermore, in vivo and in vitro metabolic studies on 1 alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyzed six-step monooxygenation to convert 1 alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1 alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is highly responsible for the metabolism of both 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3. In addition, we have succeeded in the construction of mitochondrial P450 electron transport chain consisting of ADR, ADX and each of CYP27B1 and CYP24 in E. coli cells. The coexpression system with CYP27B1 might be useful as a bioreactor to produce 1 alpha,25-dihydroxyvitamin D3. In contrast, the coexpression system with CYP24 would be applied to metabolic studies of vitamin D analogs used as drugs.
Inouye
K
K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. inouye@kais.kyoto-u.ac.jp
Sakaki
T
T
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D000325:Adrenodoxin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001709:Biotechnology; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014807:Vitamin D / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D011994:Recombinant Proteins; D000325:Adrenodoxin; D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D005287:Ferredoxin-NADP Reductase