Osami
Shoji
*a,
Takashi
Fujishiro
a,
Kousuke
Nishio
a,
Yukiko
Kano
a,
Hiroshi
Kimoto
a,
Shih-Cheng
Chien
a,
Hiroki
Onoda
a,
Atsushi
Muramatsu
a,
Shota
Tanaka
a,
Ayumi
Hori
a,
Hiroshi
Sugimoto
b,
Yoshitsugu
Shiro
b and
Yoshihito
Watanabe
*c
aDepartment of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp
bRIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan
cResearch Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. E-mail: p47297a@nucc.cc.nagoya-u.ac.jp
First published on 22nd April 2016
A substrate-binding-state mimic of H2O2-dependent cytochrome P450 that is able to catalyze monooxygenation of non-native substrates was constructed by one-point mutagenesis of P450SPα (CYP152B1). P450SPα, a long-alkyl-chain fatty acid hydroxylase, lacks any general acid–base residue around the heme. The carboxylate group of a fatty acid is thus indispensable for the generation of active species using H2O2. We prepared an A245E mutant to mimic a substrate-binding state by placing a carboxylate group at the active site. The active site structure of the A245E mutant is similar to that of the fatty-acid-bound state of P450SPα and catalyzes styrene oxidation at a rate of 280 min−1 (kcat), whereas the wild-type enzyme does not show any catalytic activity. More importantly, the same mutation, i.e. the mutation of the highly conserved threonine in P450s to glutamic acid, was also effective in introducing peroxygenase activity into P450BM3, P450cam, and CYP119. These results indicate that a variety of peroxygenases based on P450s can be constructed by one-point mutagenesis.
![]() | ||
Fig. 2 Proposed catalytic reaction mechanisms for hydroxylation of long-alkyl-chain fatty acids by wild-type P450SPα (a) and for oxidation of styrene by A245E (b). |
k cat/min−1 | K m/mM | k cat/Km/M−1 s−1 | SO![]() ![]() |
%eeb | |
---|---|---|---|---|---|
a Reaction conditions: 0.5–3 mM styrene, 4 mM H2O2 and 1 μM P450SPα in 0.1 M potassium phosphate buffer (pH 7.0) at 25 °C for 1 min. b The values under the conditions of 3 mM styrene. n.d. = not detected. | |||||
Wild-type | n.d. | ||||
A245E | 280 ± 40 | 1.5 ± 0.4 | 190 | 70![]() ![]() |
20 (S) |
A245D | 72 ± 6 | 5.3 ± 0.6 | 14 | 81![]() ![]() |
41 (S) |
A245H | 18 ± 8 | 3.5 ± 2.5 | 5.3 | 79![]() ![]() |
0.5 (S) |
R241E | n.d. |
![]() | ||
Fig. 4 The active site structures of P450SPα mutants and AaeAPO (PDB code 2YP1). a) A245E, b) A245H, c) R241E, and d) AaeAPO. Two alternative conformations of His-245 of A245H are shown. |
![]() | ||
Fig. 5 The active site structures of P450SPα: a) wild type, b) A245E, and c) R241E. The water molecule in A245E (W4) would serve as a general acid–base catalyst. |
k cat/min−1 | K m/mM | k cat/Km/M−1 s−1 | SO![]() ![]() |
%eeb | |
---|---|---|---|---|---|
a Reaction conditions: 0–15 mM styrene, 60 mM H2O2 and 5 μM P450 in 0.1 M potassium phosphate buffer (pH 7.0) at 25 °C for 1 min. b The values under the conditions of 5 mM styrene. The initial turnover rate at 70 °C is shown for CYP119. | |||||
P450BM3 T268E | 110 ± 5 | 1.5 ± 0.3 | 72 | 85![]() ![]() |
22 (R) |
P450cam T252E | 3.1 ± 0.2 | 5.4 ± 1.0 | 0.57 | 78![]() ![]() |
70 (S) |
CYP119 T213E | 16 ± 0.7 | 3.9 ± 0.5 | 4.1 | 88![]() ![]() |
29 (S) |
Footnote |
† Electronic supplementary information (ESI) available: Experimental section including expression and purification of P450s, additional figures of crystal structures, UV-vis spectra, plots of the initial turnover rate of oxidation reactions, and GC-MS analysis. Table of data collection and refinement statistics of crystal structures (PDB codes: 3VOO, 3VTJ, and 3VNO). See DOI: 10.1039/c6cy00630b |
This journal is © The Royal Society of Chemistry 2016 |