Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES

Biochemical and Biophysical Research Communications
Eriko UchidaKuniyo Inouye

Abstract

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.

References

Apr 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·M F HolickH F DeLuca
Sep 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·I T BoyleH F DeLuca
Mar 15, 1982·Biochemical and Biophysical Research Communications·A HiwatashiY Ichikawa
Dec 5, 1998·American Journal of Human Genetics·J T WangW L Miller
Jul 13, 2000·Biochemical and Biophysical Research Communications·N SawadaK Inouye
Sep 15, 2001·Archives of Biochemistry and Biophysics·N Kagawa, Q Cao
Jun 23, 2004·Biochemical and Biophysical Research Communications·Natsumi SawadaKuniyo Inouye
Jul 1, 2004·The Journal of Steroid Biochemistry and Molecular Biology·Keiko YamamotoSachiko Yamada
Nov 24, 2004·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Asja NürnbergerGerd Luippold
Mar 25, 2009·Allergology International : Official Journal of the Japanese Society of Allergology·Toshio SoneKohsuke Kino

❮ Previous
Next ❯

Citations

Dec 22, 2009·Applied Microbiology and Biotechnology·Elke BrüsehaberUwe T Bornscheuer
Apr 4, 2009·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Yoshiaki YasutakeTomohiro Tamura
Jun 18, 2010·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Edith K Y TangRobert C Tuckey
Mar 10, 2010·Biochemistry. Biokhimii︠a︡·L A NovikovaV M Shkumatov
Jan 29, 2009·Microbial Cell Factories·Olga KolajJ Gerard Wall
Mar 5, 2013·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Edith K Y TangRobert C Tuckey
Jul 27, 2010·Biochimica Et Biophysica Acta·Toshiyuki SakakiYoshitsugu Shiro
Mar 3, 2010·The Journal of Steroid Biochemistry and Molecular Biology·Edith K Y TangRobert C Tuckey
May 20, 2009·Biochemical and Biophysical Research Communications·Yoshikazu FujiiTomohiro Tamura
Feb 9, 2008·Current Opinion in Chemical Biology·Mattijs K JulsingAndreas Schmid
Oct 7, 2004·Biochemical and Biophysical Research Communications·Raku ShinkyoKuniyo Inouye
Apr 7, 2006·Biochemical and Biophysical Research Communications·Miharu AraseNorio Kagawa
Nov 29, 2005·Protein Expression and Purification·Maori Mitsuda, Masahiko Iwasaki
Nov 26, 2013·The Journal of Steroid Biochemistry and Molecular Biology·Kaori Endo-UmedaMakoto Makishima
Jun 8, 2007·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Naoko UrushinoToshiyuki Sakaki
Mar 15, 2005·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Daisuke AbeKuniyo Inouye
Dec 20, 2005·Archives of Biochemistry and Biophysics·Zhong-Liu WuF Peter Guengerich

❮ Previous
Next ❯

Related Concepts

Related Feeds

Aminoglycosides

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Aminoglycosides (ASM)

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.