Biomaterial Database

Enzymatic studies on the key enzymes of vitamin D metabolism; 1 alpha-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24). 2001

K Inouye, and T Sakaki

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. inouye@kais.kyoto-u.ac.jp

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D003577	Cytochrome P-450 Enzyme System	A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism.	Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D005287	Ferredoxin-NADP Reductase	An enzyme that catalyzes the oxidation and reduction of FERREDOXIN or ADRENODOXIN in the presence of NADP. EC 1.18.1.2 was formerly listed as EC 1.6.7.1 and EC 1.6.99.4.	Adrenodoxin Reductase,Iron-Sulfur Protein Reductase,NADPH-Ferredoxin Reductase,Ferredoxin NADP Reductase,Iron Sulfur Protein Reductase,NADPH Ferredoxin Reductase,Protein Reductase, Iron-Sulfur,Reductase, Adrenodoxin,Reductase, Ferredoxin-NADP,Reductase, Iron-Sulfur Protein,Reductase, NADPH-Ferredoxin
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000325	Adrenodoxin	An iron-sulfur protein which serves as an electron carrier in enzymatic steroid hydroxylation reactions in adrenal cortex mitochondria. The electron transport system which catalyzes this reaction consists of adrenodoxin reductase, NADP, adrenodoxin, and cytochrome P-450.
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001709	Biotechnology	Body of knowledge related to the use of organisms, cells or cell-derived constituents for the purpose of developing products which are technically, scientifically and clinically useful. Alteration of biologic function at the molecular level (i.e., GENETIC ENGINEERING) is a central focus; laboratory methods used include TRANSFECTION and CLONING technologies, sequence and structure analysis algorithms, computer databases, and gene and protein structure function analysis and prediction.	Biotechnologies
D013250	Steroid Hydroxylases	Cytochrome P-450 monooxygenases (MIXED FUNCTION OXYGENASES) that are important in steroid biosynthesis and metabolism.	Steroid Hydroxylase,Steroid Monooxygenases,Hydroxylase, Steroid,Hydroxylases, Steroid,Monooxygenases, Steroid