Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System
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 cr...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-01-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/3/1837 |
| Summary: | 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 <i>c</i> and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme <i>c</i> containing an electron transfer protein derived from <i>Rhizobium radiobacter</i> (CYTc) was selected to prepare SpyCatcher-fused heme <i>c</i>. 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 <span style="font-variant: small-caps;">d</span>-amino acid oxidase (DAAOx), and an engineered FMN-dependent <span style="font-variant: small-caps;">l</span>-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. |
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| ISSN: | 1661-6596 1422-0067 |