Interaction between mitochondrial CYP27B1 and adrenodoxin: role of arginine 458 of mouse CYP27B1.
A molecular modeling study of CYP27B1 suggests that Arg458 of mouse CYP27B1 is involved in interaction with adrenodoxin (ADX). Thus, we generated CYP27B1 mutants R458K and R458Q and revealed their enzymatic properties. Substrate-induced difference spectra and K(m) values for 1alpha-hydroxylation of 25(OH)D3 indicate that the replacement of Arg458 with Lys or Gln does not affect substrate binding. However, these mutants showed remarkable decreases of both kcat values and the ratio of product formation to NADPH oxidation (coupling efficiency). A high K(m) value of R458Q for ADX concentration and a decrease of rate constant of the first electron transfer seem reasonable considering that the conversion from Arg to noncharged Gln abolishes salt-bridge formation with the acidic residue of ADX. On the other hand, R458K showed atypical kinetics for ADX concentration with Hill's constant of 2.0 and high catalytic activity at high ADX concentration by increase of coupling efficiency. These results suggest that conformational change of R458K by binding the two ADX molecules is essential for 1alpha-hydroxylation of 25(OH)D3. On the other hand, binding one ADX molecule is sufficient for the conformational change of the wild-type CYP27B1, judging from its Michaelis-Menten-type kinetics for ADX concentration with high coupling efficiency. These results suggest that ADX functions as an effector for the oxygen transfer reaction in addition to being an electron donor for CYP27B1.
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Naoko
N
Urushino
Keiko
K
Yamamoto
Norio
N
Kagawa
Shinichi
S
Ikushiro
Masaki
M
Kamakura
Sachiko
S
Yamada
Shigeaki
S
Kato
Kuniyo
K
Inouye
Toshiyuki
T
Sakaki
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D000325:Adrenodoxin / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000502:physiology*; D002112:Calcifediol / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D016415:Sequence Alignment; D013053:Spectrophotometry
D000325:Adrenodoxin; D009249:NADP; D001120:Arginine; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D005287:Ferredoxin-NADP Reductase; D002112:Calcifediol