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METABOLISM OF VITAMIN D ANALOGS BY HUMAN ENZYMES

Research Project

Project/Area Number 13660092
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

SAKAKI Toshiyuki  AGRICULTURE, ASSOCIATE PROFFESOR, 農学研究科, 助教授 (70293909)

Co-Investigator(Kenkyū-buntansha) INOUYE Kuniyo  AGRICULTURE, PROFFESSOR, 農学研究科, 教授 (10223249)
Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
KeywordsVITAMIN D / CYTOCHROME P450 / E. COLI EXPRESSION SYSTEM / VITAMIN D HYDROXYLASE / PREDICTION OF METABOLISM / SITE-DIRECTED MUTAGENESIS / 部位特異的変異
Research Abstract

We have determined eight types of missense mutants of CYP27B1 from vitamin D-dependent rickets type I patients, and examined their enzymatic properties. In addition, we generated various mutants of CYP27B1 and CYP27A1 to examine the roles of the mutated amino acid residues of the CYP27B1 mutants. The experimental data strongly suggested that they were involved in supply of protons, heme-propionate binding, activation of oxygen, substrate-binding, and stabilization of tertiary structure. Based on these results, the tertiary structures of CYP27B1 and CYP27A1 were constructed by computer modeling.
On CYP24-dependent metabolism of three vitamin D analogs described below, clear species-based difference was observed between humans and rats. The recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of vitamin D analogs in human target tissues before clinical trials. When F_6-1α,25(OH)_2D_3 was added to the recombinant E. coli cells expressing human CYP24, the putative ether compound with no affinity for vitamin D receptor (VDR) was observed. However, this metabolite was not observed in those expressing rat CYP24. On the metabolism of maxacalcitol, human CYP24 cleaved the O_<22>-C_<23> bond at the first step of the metabolism, while rat CYP24 cleaved it at the third step, suggesting rapid inactivation of maxacalcitol in human body. In the case of the metabolism of 1α,25(OH)_2-20-epi-D_3, rat CYP24 converted 1α,25(OH)_2-20-epi-D_3 to 25.26,27-trinor-lα (OH)-24(COOH) indicating completely inactivation of 1α,25(OH)_2-20-epi-D_3. On the other hand, human CYP24 mainly converted 1α,25(OH)_2-20-epi-D_3 to 1α,23,25(OH)_3-24-oxo-20-epi-D_3 which retains the affinity for VDR.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Sawada, N., Sakaki, T., Kitanaka, S., Kato, S., Inouye, K: "Structure-function analysis of CYP27B1 and CYP27A1"Eur. J. Biochem.. 268. 6607-6615 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Inouye, K., Sakaki, T.: "Enzymatic studies on the key enzymes of vitamin D metabolism ; 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24)"Biotech. Annu. Rev.. 7. 179-194 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 榊 利之, 井上國世: "ビタミンD_3の代謝を司る3つの酵素"化学と生物. 39・11. 705-707 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Sasaki T, Sawada N, Abe D, Komai K, Okano T, Ohta M, Inouye K他: "Metabolism of 26,26,26,27,27,27-F_6-1α,25-dihydroxyvitamin D_3 by CYP24 : Species-based difference between humans and rats"Biochem. Pharmacol.. (発表予定). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Natsumi Sawada, Toshiyuki Sakaki, Sachiko Kitanaka, Shigeaki Kato and Kuniyo Inouye: "Structure-function analysis of CYP27B1 and CYP27A1: Studies on mutants from patients with vitamin D-dependent rickets type I (VDDR-I) and cerenbrotendinous xanthomatosis (CTX)"Eur. J. Biochem.. 268. 6607-6615 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Kuniyo Inouye and Toshiyuki Sakaki: "Enzymatic studies on the key enzymes of vitamin D metabolism; 1-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24)"Biothchnol. Ann. Rev.. 7. 179-194 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Kuniyo Inouye and Toshiyuki Sakaki: "Three enzymes for vitamin D_3 metabolism (In Japanese)"Kagaku to Seibutsu. 39. 705-707 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Toshiyuki Sakaki, Natsumi Sawada, Daisuke Abe, Koichiro Komai, Shunichi Shiozawa, Yasuki Nonaka, Kimie Nakagawa, Toshio Okano, Miho Ohta and Kuniyo Inouye.: "Metabolism of 26,26,26,27,27,27-F_6-lα,25-dihydroxyvitamin D_3 by CYP24: Species based difference between humans and rats"Biochem. Pharmacol.. (in press). (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Sakaki T, Sawada N, Abe D, Komai K, Okano T, Ohta M, Inouye K 他: "Metabolism of 26,26,26,27,27,27-F_6-1α, 25-dihydroxyvitamin D_3 by CYP24 ; Species-based difference between humans and rats"Biochem. Pharmacol.. (発売予定). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Sawada, N., Sakaki, T., Kitanaka, S., Kato, S., Inouye, K.: "Structure-function analysis of CYP27B1 and CYP27A1"Eur. J. Biochem.. 268. 6607-6615 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Inouye, K., Sakaki, T.: "Enzymatic studies on the key enzymes of vitamin D metabolism;1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24)"Biotech. Annu. Rev.. 7. 179-194 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 榊 利之, 井上國世: "ビタミンD_3の代謝を司る3つの酵素"化学と生物. 39・11. 705-707 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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