私たちは最新の遺伝子工学技術で健康の維持・増進に繋がる機能性食品の開発に役立つ研究をしています。
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Structure and function of UDP-glucuronosyltransferase and its application for xennobiotics.
Regulation of developmental signals regarding morphogenesis in animals.
Laboratory of Food Science and Technology,
Department of Biotechnology, Toyama Prefectural University,
Kurokawa 5180, Imizu, Toyama 939-0398, JAPAN
TEL: +81-766-56-7500
FAX: +81-766-56-2498
E-mail:
tsakaki_AT_pu-toyama.ac.jp (T.Sakaki)
ikushiro_AT_pu-toyama.ac.jp (S.Ikushiro)
kamakura_AT_pu-toyama.ac.jp (M. Kamakura)
PLEASE REPLACE '_AT_' WITH '@'
The vitamin D3 25-hydroxylases (CYP27A1 and CYP2R1), 25-hydroxyvitamin D3 1 α-hydroxylase (CYP27B1) and 1α,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli (mitochondrial CYP27A1, CYP27B1, and CYP24A1) or S. cerevisiae (microsomal CYP2R1), enzymatic properties of the P450s were examined in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP2R1 catalyzed 25-hydroxylation of vitamin D3, but CYP27A1 produced at least seven minor metabolites including 1 α,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1 α position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1 α-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1α-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1α,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1α,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1α,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1, and CYP24A1.
Vitamin D analogs are potentially useful for clinical treatments of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer. On the use of vitamin D analogs, the information of metabolism in target tissues such as kidney, small intestine, and bones is pharmacologically essential. CYP24A1 is considered to be associated with the major metabolic pathways of the vitamin D analogs in these tissues. Species-based difference on the metabolism of vitamin D3 analogs appears to be originated from the difference of CYP24A1-dependent reactions. Since human kidney specimen is not easily obtained and substrate specificities of CYP24A1 vary among species, the development of an in vitro system containing human CYP24A1 was essential for the prediction of the drug metabolism in human kidney. We examined CYP24A1-dependent metabolism of such vitamin D analogs as A-ring diastereomers and 20-epimer of 1α,25(OH)2D3, 2α-propoxy- 1α,25(OH)2D3 2α-(3-hydroxypropoxy)- 1α,25(OH)2D3, and 26,26,26,27,27,27- F6-1α,25(OH)2D3. Good correlation was observed between increasing effect on serum calcium level in rats and kcat/Km value of CYP24A1 for each of the vitamin D analogs. In addition, species-based difference was also observed in CYP24A1-dependent metabolism of vitamin D analogs between humans and rats. These results strongly suggest the usefulness of the recombinant systems harboring CYP24A1 for predicting the metabolism and efficacy of vitamin D analogs before clinical trials.
Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) was examined using a recombinant enzyme system and human liver microsomes. A good correlation (R = 0.92) was observed between 2,3,7-triCDD 8-hydroxylation and phenacetin O-deethylation in human liver microsomes, suggesting that CYP1A2 is responsible for 2,3,7-triCDD 8-hydroxylation in human livers. On the other hand, multiple human UGT isozymes showed glucuronidation activity toward 8-hydroxy-2,3,7-triCDD (8-OH-2,3,7-triCDD). Of these UGTs, UGT1A1, 1A9, and 2B7, which are constitutively expressed in human livers, showed remarkable activity toward 8-OH-2,3,7-triCDD. These results strongly suggest that 2,3,7-triCDD would be firstly converted to 8-OH-2,3,7-triCDD by CYP1A2, then further converted to its glucuronide by UGT1A1, 1A9, and 2B7 in human liver.
Our previous studies revealed that mono-, di-, and tri-chloro-dibenzo-p-dioxins were good substrates of rat CYP1A1. However, rat CYP1A1 showed no activity toward 2,3,7,8-TCDD. Based on these results, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among the mutants examined, F240A showed a conversion of 2,3,7,8-TCDD to 8-hydroxy-2,3,7-triCDD. To our best knowledge, the F240A mutant of rat CYP1A1 is the first enzyme to be verified as a 2,3,7,8-TCDD-metabolizing enzyme. In addition, we successfully expressed N-terminal truncated F240A mutant (ΔF240A) in E. coli cells. These results suggest possible application of fungal or prokaryotic cells expressing F240A or ΔF240A to the bioremediation of PCDD- contaminated soil.
We examined metabolism of sesamin by cytochrom P450 (P450) using yeast expression system and human liver microsomes, and found that CYP2C9 was the most important P450 isoform for sesamin catecolization in human liver. We also found a weak mechanism-based inhibition of CYP2C9 by sesamin. Next step, we focused on the metabolism of sesamin monocatechol that was further converted into the dicatechol form by cytochrome P450 or the glucuronide by UDP-glucuronosyltransferase (UGT). Catecholization of sesamin monocatechol enhances its anti-oxidant activity, whereas glucuronidation strongly reduces its anti-oxidant activity. In human liver microsomes, the glucuronidation activity was much higher than the catecholization activity toward sesamin monocatechol. Kinetic studies using recombinant human UGT isoforms identified UGT2B7 as the most important UGT isoform for glucuronidation of sesamin monocatechol.
We also observed the methylation activity toward sesamin monocatechol by catechol O-methyl transferase (COMT) in human liver cytosol. Based on these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in the human liver.
CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D3 (VD3) to its active form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by a two-step hydroxylation reaction. Our crystal structure analysis has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. Thus, 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 D3 hydroxylase. The double mutant R73V/R84A exhibited 435- and 110-fold higher kcat/Km values for the 25-hydroxylation of 1α-hydroxyvitamin D3 and 1α-hydroxylation of 25-hydroxyvitamin D3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD3 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 73 and 84 positions affect the location and conformation of the bound compound in the reaction site, and those in the transient binding site, respectively.
UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism of a broad range of xenobiotic and endogenous compounds. UGT catalyzes the transfer of a glucuronic acid group to a variety of functional groups on the substrates. Expression of UGT in mammals is thought to be regulated in both a tissue- and developmental-specific manner. Furthermore, induction of genes encoding UGT occurs after exposure to xenobiotics including drugs, environmental pollutants and dietary compounds. In human, isoforms of UGT1A subfamily catalyze the glucuronidation of a greater proportion of drugs, suggesting that the expression of UGT1A isoforms is responsible for the clearance of a diverse range of drugs. To analyze the expression of rat and human UGT1A isoforms, we have developed polyclonal antibodies against specific peptide regions within the isoforms (UGT1A1, 1A3, 1A4, 1A6 and 1A9) and C-terminal common region. The prepared antipeptide antibodies were found to be highly monospecific for each UGT1A isoform and no cross-reactivity with UGT2B isoforms was detected. Analysis of UGT1A protein levels in human hepatic microsomes using these antibodies demonstrated interindividual differential expression of each isoform. The range of UGT1A and UGT1A1 content was 4.5 and 4.8-fold, respectively. These monospecific antipeptide antibodies against human UGT1A isoforms provide an important tool to analyze tissue distribution and interindividual expression levels of UGT1As.
In addition to drugs and environmental pollutants, dietary compounds including plant secondary metabolites are glucuronidated by UGT isoforms and its biological effects such as antioxidant capacity depend, in partly, on formation of the glucuronides. To evaluate the contribution of each UGT isoforms responsible for glucuronidation towards one of the dietary bioflavonoid, quercetin, we have constructed heterologus expression system of rat and human UGT1A and UGT2B isoforms ,and analyzed the glucuronidation using the recombinant UGTs. Formation of quercetin glucuronides was analyzed by UV-HPLC using C30 reverse phase column. Rat and human UGT1A1 catalyzed glucuronidation towards 3’- and 4’-hydroxyl moiety of B-ring in quercetin with high catalytic efficiency and substrate inhibition kinetics. In contrast, both UGT1A6 were unable to catalyze the glucuronidation of any hydroxyl moiety. In human, UGT1A9 produced 3’-, 4’-, 3- and 7-glucuronides. Most UGT2B isoforms in rat and human did not catalyze the quercetin glucuronidation. UGT2B1 in rat and UGT2B7 in human have a moderate activity for quercetin. Extrahepatic human UGT1A7, UGT1A8 and UGT1A10, which are found throughout the gastrointestinal tract, show the high glucuronidation efficiency towards 3-, 3’- and 4’-hydroxyl moiety, respectively. These isoforms have a regioselectivity of glucuronidating site in quercetin in spite of highly similarity of amino acid sequences. These results suggest that glucuronidation towards quercetin are catalyzed by several UGT isoforms, and recombinant rat and human UGT expression system provides information on the regioselectivity of glucuronidation of dietary bioflavonoids.
Glucuronidation, which is catalyzed by isoforms of UDP-glucuronosyltransferases (UGT), is the most common pathway for detoxification and elimination of hydrophobic xenobiotics occurring in tissues of most mammals. Because of their ubiquitous nature and high physiological significance, development of an efficient in vitro synthesis of glucuronides often becomes critical during studies of drug metabolism undertaken in the development of a new pharmaceutical product. Glucuronides have been obtained by chemical synthesis, by tedious isolation and purification of in vivo metabolites from biological samples of experimental animals, and by biosynthesis using hepatic microsomes as enzyme source. In order to synthesize the glucuronides as drug metabolites, we have now developed several mammalian CYP, UGT and UDP-glucose dehydrogenase (UGDH) coexpression systems in budding yeast.
We have previously constructed the expression system of several mammalian CYP and UGT isoforms in budding yeast cells, Saccharomyces cerevisiae AH22, to analyze the structure and function of these xenobiotic metabolizing enzymes. Yeast cells lack the ability of production of UDP-glucuronic acid (UDP-GlcUA) from UDP-glucose to synthesize the glucuronide in whole cells. To achieve the production of UDP-GlcUA for co-substrate of glucuronidation, UGDH gene deriver from rat or plant was introduced to yeast cell. Most glucuronide was found in reaction medium with time-dependent production, suggesting the functional expression of both enzymes and the presence of endogenous transport system for glucuronide in yeast. Optimization of the reaction conditions resulted in 95% conversion of 7-hydroxycoumarine(7HC) into its glucuronide. Compared with fission yeast system as host cell , budding yeast appears to be more competent for glucuronide formation. Mycophenolic acid with multiple glucuronidating sites was conjugated as UGT isoform-dependent formation, suggesting that the regiospecific glucuronides of several drugs could be obtained using UGT1A and 2B isoforms. In order to synthesize glucuronide from CYP-dependent metabolite during Phase I and II processes, rat CYP1A1 and yeast NADPH-P450 reductase were coexpressed with UGT in yeast. The resultant recombinant yeast cells with xenobiotic metabolizing enzymes was able to produce directly the glucuronide from 7-ethoxycoumarin via 7HC. This coexpression system of mammalian CYP, UGT and UGDH in budding yeast would be a powerful tool for enzyme-assisted synthesis of various xenobiotic metabolites including glucuronides.
The honeybee (Apis mellifera) forms two female castes, the queen and the worker. This dimorphism depends not on genetic differences, but on ingestion of royal jelly (RJ), though the mechanism through which RJ regulates caste differentiation has long remained unknown. Here I show that a 57-kDa protein in RJ, designated as royalactin in a previous report, induces differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybee. Surprisingly, it also showed similar effects in fruit fly (Drosophila melanogaster). Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase (MAP kinase), which was involved in the decrease of developmental time, and increased the titer of juvenile hormone (JH), an essential hormone for ovary development. These actions were mediated by epidermal growth factor receptor (Egfr) in fat body of both insects, because knockdown of Egfr expression resulted in a defect of all phenotypes induced by royalactin. Thus, these findings indicate that a specific factor in RJ, royalactin, drives queen development through an Egfr-mediated signaling pathway.
The environmental cues during developmental period influence animal cell growth and histogenesis to change phenotypes of individuals. In this study, we investigate how intracellular signaling pathways, juvenile hormone-regulating signals and ecdysone receptor signalling factors control body size and morphogenesis in Drosophila and honeybees.
Ikushiro S, Nishikawa M, Masuyama Y, Shouji T, Fujii M, Hamada M, Nakajima N, Finel M, Yasuda K, Kamakura M, Sakaki T.
Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae.
Mol Pharm. 13, 2274-2282 (2016)
Mise S, Haga Y, Itoh T, Kato A, Fukuda I, Goto E, Yamamoto K, Yabu M, Matsumura C, Nakano T, Sakaki T, Inui H.
Structural Determinants of the Position of 2,3',4,4',5-Pentachlorobiphenyl (CB118) Hydroxylation by Mammalian Cytochrome P450 Monooxygenases.
Toxicol Sci. pii: kfw086 (2016)
Hayashi K, Yasuda K, Yogo Y, Takita T, Yasukawa K, Ohta M, Kamakura M, Ikushiro S, Sakaki T.
Sequential hydroxylation of vitamin D2 by a genetically engineered CYP105A1.
Biochem Biophys Res Commun. 473, 853-858 (2016)
Corcoran A, Nadkarni S, Yasuda K, Sakaki T, Brown G, Kutner A, Marcinkowska E.
Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D2 as Potential Anti-Leukemic Agents.
Int J Mol Sci. 17, e91 (2016)
Munetsuna E, Kittaka A, Chen TC, Sakaki T.
Metabolism and Action of 25-Hydroxy-19-nor-Vitamin D3 in Human Prostate Cells.
Vitam Horm. 100, 357-377 (2016)
Nakamura T, Miyoshi N, IshiiT, , Nishikawa M, Ikushiro S, Watanabe T.
Activation of transient receptor potential ankyrin 1 by quercetin and its analogs.
Biosci Biotechnol Biochem. 80, 949-954 (2016)
(Noriyuki Kasai)
""Functional analysis of cytochoromes P450 derived from the white fungus Phanerochaete chrysosporium"" (2010)
(Naoko Urushino)
""Structure-function analysis of mammalian vitamin D hydroxylases"" (2010)
(Shunsuke Uchihashi)
""Structure-function analysis and gene expression of mammalian UDP-glucuronosyltransferase"" (2013)
(Kaori Yasuda)
""Prediction of the metabolism of drug and food factors by drug metabolizing enzymes in human"" (2013)
(Maya Kamao)
""Studies om vitamin D level in biological samples and novel metabolites of vitamin D"" (2016)
