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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Catalysis Science and Technology  

Cytochrome P450 peroxygenases belonging to the CYP152 family catalyse the oxidation of fatty acids using H2O2. CYP152N1 isolated from Exiguobacterium sp. AT1b exclusively catalyses the α-selective hydroxylation of myristic acid at physiological H2O2 concentration. However, a series of shorter-alkyl-chain fatty acids such as tridecanoic acid were produced from myristic acid by increasing the concentration of H2O2 (1-10 mM). The yield of tridecanoic acid from myristic acid reached 17%. An 18O-labeled oxidant study suggested that CYP152N1 catalysed the overoxidation of α-hydroxymyristic acid to form α-ketomyristic acid, which in turn was spontaneously decomposed by H2O2 to yield tridecanoic acid. Crystal structure analysis of CYP152N1 revealed its high similarity to other CYP152 family enzymes, such as CYP152A1 and CYP152B1. MD simulations of α-hydroxymyristic acid accommodated in CYP152N1 proposed a possible pre-oxidation conformation of α-hydroxymyristic acid for the decarboxylation reaction.

DOI： 10.1039/C7CY02263H

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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Dalton Transactions  

Cytochrome P450<inf>SPα</inf> (P450<inf>SPα</inf>) and cytochrome P450<inf>BSβ</inf> (P450<inf>BSβ</inf>) belonging to the CYP152 family of enzymes (CYP152s) can utilize H<inf>2</inf>O<inf>2</inf> efficiently as an oxidant for the generation of compound I. Although P450<inf>SPα</inf> and P450<inf>BSβ</inf> have very high substrate specificity and catalyse hydroxylation of long-chain fatty acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including medium chain length n-alkyl carboxylic acids as "decoy molecules." Although we had assumed that acetic acid did not serve as a decoy molecule, P450<inf>SPα</inf> and P450<inf>BSβ</inf> efficiently catalysed oxidation of non-native substrates when the reaction was carried out at a high concentration of acetate anion. The turnover rate for epoxidation of styrene catalysed by P450<inf>BSβ</inf> in the presence of 1 M acetate anion reached 590 ± 30 min^-1.

DOI： 10.1039/c5dt00797f

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Journal of Molecular Sciences  

X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal’s domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method.

DOI： 10.3390/ijms242316640

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACS Catalysis  

Biological methane oxidation is a highly desirable method for the conversion of natural gas into a liquid to meet the increasing demand for fuel and chemical feedstock as well as reducing the potent greenhouse effects of methane emissions. Because natural hemoenzymes that can catalyze the conversion of methane to methanol have not been found, it has long been considered that hemoenzymes, including cytochrome P450s (P450s), cannot catalyze the oxidative conversion of methane. Herein, we report the catalytic oxidation of methane by wild-type P450BM3, without any mutagenesis, in the presence of chemically evolved dummy substrates (decoy molecules) under high-pressure methane at 10 MPa. Our studies showed that methane was catalytically converted into methanol at room temperature with a total turnover number of 4.

DOI： 10.1021/acscatal.3c01158

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/nar/gkac1082

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ChemBioChem  

Tetraphenylporphyrin (TPP) is a symmetrically substituted synthetic porphyrin whose properties can be readily modified, providing it with significant advantages over naturally occurring porphyrins. Herein, we report the first example of a stable complex between a native biomolecule, the haemoprotein HasA, and TPP as well as its derivatives. The X-ray crystal structures of nine different HasA-TPP complexes were solved at high resolutions. HasA capturing TPP derivatives was also demonstrated to inhibit growth of the opportunistic pathogen Pseudomonas aeruginosa. Mutant variants of HasA binding FeTPP were shown to possess a different mode of coordination, permitting the cyclopropanation of styrene.

DOI： 10.1002/cbic.202200095

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/jb/mvab087

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACS Catalysis  

Highly effective dipeptidic decoy molecules, which stimulate the direct hydroxylation of benzene by wild-type cytochrome P450BM3, were successfully developed through a rationally designed screening method. Extensive synthesis and step-wise screening of over 600 dipeptide derivatives were performed for the efficient evolution of decoy molecules. In the presence of N-(3-cyclopentyl)propanoyl-l-pipecolyl-l-phenylalanine (3CPPA-Pip-Phe), one of the most effective decoy molecules discovered herein, the catalytic turnover frequency and total turnover number for benzene hydroxylation reached 405 min-1 P450BM3-1 and 54,500 P450BM3-1, respectively. Furthermore, the decoy molecules developed in this work drastically accelerated the hydroxylation of other non-native substrates, such as anisole and toluene, as well as nonaromatic compounds, such as cyclohexane, propane, and ethane. Using N-enanthoyl-l-pipecolyl-l-phenylalanine (C7AM-Pip-Phe), the hydroxylation rate for ethane to ethanol reached 82.7 min-1 P450BM3-1.

DOI： 10.1021/acscatal.0c01951

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Chemical Communications  

We prepared enzyme-immobilized hydrogels and investigated the effects of the cross-linking density and polymer properties on their oxidation reaction rate. The oxidation rate of enzyme-immobilized hydrogels increased as the cross-linking density in the hydrogels increased. In addition, we controlled the oxidation rate using hydrogels exhibiting an appropriate interaction with a decoy molecule in the hydrogel.

DOI： 10.1039/D0CC01332C

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Chemical Communications  

Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.

DOI： 10.1039/C8CC02760A

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Catalysis Science and Technology  

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.

DOI： 10.1039/c6cy00630b

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開催年月日： 2022年8月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2022年6月

記述言語：日本語   会議種別：口頭発表（一般）  

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開催年月日： 2022年5月

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

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開催年月日： 2022年3月

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

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開催年月日： 2021年11月

会議種別：シンポジウム・ワークショップ パネル（指名）  

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開催年月日： 2016年9月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2015年6月

記述言語：英語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2023年11月

会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

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開催年月日： 2023年1月

会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催年月日： 2023年1月

記述言語：日本語   会議種別：ポスター発表  

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開催年月日： 2022年10月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2021年12月

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開催年月日： 2018年3月

記述言語：英語   会議種別：口頭発表（一般）  

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開催年月日： 2016年6月

記述言語：日本語   会議種別：口頭発表（一般）  

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開催年月日： 2016年3月

記述言語：日本語   会議種別：口頭発表（一般）  

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