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タイトル: Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis
著者: Ogawa, Noriyuki
Kimura, Takehide
Umehara, Fumi
Katayama, Yuki
Nagai, Go
Suzuki, Keiko
Aisaka, Kazuo
Maruyama, Yukie
Itoh, Takafumi
Hashimoto, Wataru
Murata, Kousaku
Ichimura, Michio
著者名の別形: 小川, 徳之
木村, 豪秀
楳原, 芙美
片山, 有基
永井, 豪
鈴木, 恵子
相阪, 和夫
丸山, 如江
伊藤, 貴文
橋本, 渉
村田, 幸作
市村, 通朗
キーワード: Biotechnology
Oxidoreductases
発行日: 30-Jan-2019
出版者: Springer Nature
誌名: Scientific Reports
巻: 9
論文番号: 942
抄録: The currently available haemoglobin A1c (HbA1c) enzymatic assay consists of two specific steps: proteolysis of HbA1c and oxidation of the liberated fructosyl peptide by fructosyl peptide oxidase (FPOX). To develop a more convenient and high throughput assay, we devised novel protease-free assay system employing modified FPOX with HbA1c oxidation activity, namely HbA1c direct oxidase (HbA1cOX). AnFPOX-15, a modified FPOX from Aspergillus nidulans, was selected for conversion to HbA1cOX. As deduced from the crystal structure of AnFPOX-15, R61 was expected to obstruct the entrance of bulky substrates. An R61G mutant was thus constructed to open the gate at the active site. The prepared mutant exhibited significant reactivity for fructosyl hexapeptide (F-6P, N-terminal amino acids of HbA1c), and its crystal structure revealed a wider gate observed for AnFPOX-15. To improve the reactivity for F-6P, several mutagenesis approaches were performed. The ultimately generated AnFPOX-47 exhibited the highest F-6P reactivity and possessed HbA1c oxidation activity. HbA1c levels in blood samples as measured using the direct assay system using AnFPOX-47 were highly correlated with the levels measured using the conventional HPLC method. In this study, FPOX was successfully converted to HbA1cOX, which could represent a novel in vitro diagnostic modality for diabetes mellitus.
記述: 糖尿病マーカー、ヘモグロビンA1cを直接酸化できる酵素の創製 --シンプルかつ短時間測定が可能な新規測定試薬開発に大きく前進--. 京都大学プレスリリース. 2019-01-31.
著作権等: © The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
URI: http://hdl.handle.net/2433/236130
DOI(出版社版): 10.1038/s41598-018-37806-x
関連リンク: http://www.kyoto-u.ac.jp/ja/research/research_results/2018/190130_1.html
出現コレクション:学術雑誌掲載論文等

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