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Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.
Thomas M B Reichhart1,2, Stefan Scheiblbrandner1, Christoph Sygmund2
1Biocatalysis and Biosensing Laboratory, Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Protein Science : a Publication of the Protein Society
|June 14, 2023
Summary
Interface engineering improved cellobiose dehydrogenase (CDH) for biosensors by enhancing interdomain electron transfer (IET) at physiological pH. However, modifications reduced direct electron transfer (DET), requiring further optimization for bioelectronic applications.
Area of Science:
- Biocatalysis
- Bioelectrochemistry
- Protein Engineering
Background:
- Cellobiose dehydrogenase (CDH) is a key bioelectrocatalyst for direct electron transfer (DET) in biosensors and biofuel cells.
- Its acidic pH optimum and slow interdomain electron transfer (IET) at physiological pH limit its application in glucose measurements.
- Electrostatic repulsion at the interface between the dehydrogenase and cytochrome domains (CYT) hinders electron transfer.
Purpose of the Study:
- To engineer the CDH enzyme interface to accelerate IET at physiological pH.
- To investigate the mechanisms behind improved IET and identify strategies for future bioelectronic applications.
Main Methods:
- Rational interface engineering of the CYT domain by mutating acidic amino acids.
- Phylogenetic and structural analyses to guide variant design.
- Characterization of 17 CDH variants for pH optimum, IET rate, and DET.
Main Results:
- Five single mutations (G71K, D160K, Q174K, D177K, M180K) enhanced pH optimum and IET rate.
- Combinatorial variants shifted pH optimum to 7.0 and increased IET at pH 7.5 over 12-fold.
- Increased positive charges on CYT improved IET but decreased DET, indicating a trade-off.
Conclusions:
- Interface engineering is effective for optimizing CDH's pH optimum and IET.
- Electrostatic steering and hydrogen bonding stabilize the enzyme's closed state, enhancing IET.
- Maintaining DET is crucial for future bioelectronic applications of engineered CDH.
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