Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Autor: Reichhart TMB; Biocatalysis and Biosensing Laboratory, Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.; DirectSens GmbH, Klosterneuburg, Austria., Scheiblbrandner S; Biocatalysis and Biosensing Laboratory, Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria., Sygmund C; DirectSens GmbH, Klosterneuburg, Austria., Harreither W; Biocatalysis and Biosensing Laboratory, Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.; DirectSens GmbH, Klosterneuburg, Austria., Schenkenfelder J; Biocatalysis and Biosensing Laboratory, Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria., Schulz C; DirectSens GmbH, Klosterneuburg, Austria., Felice AKG; Biocatalysis and Biosensing Laboratory, Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.; DirectSens GmbH, Klosterneuburg, Austria., Gorton L; Department of Analytical Chemistry/Biochemistry, Lund University, Lund, Sweden., Ludwig R; Biocatalysis and Biosensing Laboratory, Institute of Food Technology, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2023 Aug; Vol. 32 (8), pp. e4702.
DOI: 10.1002/pro.4702
Abstrakt: Cellobiose dehydrogenase (CDH) is a bioelectrocatalyst that enables direct electron transfer (DET) in biosensors and biofuel cells. The application of this bidomain hemoflavoenzyme for physiological glucose measurements is limited by its acidic pH optimum and slow interdomain electron transfer (IET) at pH 7.5. The reason for this rate-limiting electron transfer step is electrostatic repulsion at the interface between the catalytic dehydrogenase domain and the electron mediating cytochrome domain (CYT). We applied rational interface engineering to accelerate the IET for the pH prevailing in blood or interstitial fluid. Phylogenetic and structural analyses guided the design of 17 variants in which acidic amino acids were mutated at the CYT domain. Five mutations (G71K, D160K, Q174K, D177K, M180K) increased the pH optimum and IET rate. Structure-based analysis of the variants suggested two mechanisms explaining the improvements: electrostatic steering and stabilization of the closed state by hydrogen bonding. Combining the mutations into six combinatorial variants with up to five mutations shifted the pH optimum from 4.5 to 7.0 and increased the IET at pH 7.5 over 12-fold from 0.1 to 1.24 s -1 . While the mutants sustained a high enzymatic activity and even surpassed the IET of the wild-type enzyme, the accumulated positive charges on the CYT domain decreased DET, highlighting the importance of CYT for IET and DET. This study shows that interface engineering is an effective strategy to shift the pH optimum and improve the IET of CDH, but future work needs to maintain the DET of the CYT domain for bioelectronic applications.
(© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
Databáze: MEDLINE
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