Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System

Takumi Yanase; Junko Okuda-Shimazaki; Ryutaro Asano; Kazunori Ikebukuro; Koji Sode; Wakako Tsugawa

doi:10.3390/ijms24031837

International Journal of Molecular Sciences (Jan 2023)

Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System

Takumi Yanase,
Junko Okuda-Shimazaki,
Ryutaro Asano,
Kazunori Ikebukuro,
Koji Sode,
Wakako Tsugawa

Affiliations

Takumi Yanase: Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA
Junko Okuda-Shimazaki: Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA
Ryutaro Asano: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei 184-8588, Tokyo, Japan
Kazunori Ikebukuro: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei 184-8588, Tokyo, Japan
Koji Sode: Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA
Wakako Tsugawa: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei 184-8588, Tokyo, Japan

DOI: https://doi.org/10.3390/ijms24031837
Journal volume & issue: Vol. 24, no. 3
p. 1837

Abstract

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The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method.

Published in International Journal of Molecular Sciences

ISSN: 1661-6596 (Print); 1422-0067 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Biology (General); Science: Chemistry
Website: http://www.mdpi.com/journal/ijms

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