Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Thomas M B Reichhart, Stefan Scheiblbrandner, Christoph Sygmund, Wolfgang Harreither, Josef Schenkenfelder, Christopher Schulz, Alfons K G Felice, Lo Gorton, Roland Ludwig

Protein science : a publication of the Protein Society 2023 Aug

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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.

Citation

Thomas M B Reichhart, Stefan Scheiblbrandner, Christoph Sygmund, Wolfgang Harreither, Josef Schenkenfelder, Christopher Schulz, Alfons K G Felice, Lo Gorton, Roland Ludwig. Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer. Protein science : a publication of the Protein Society. 2023 Aug;32(8):e4702