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Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer

Reichhart, Thomas M.B. ; Scheiblbrandner, Stefan ; Sygmund, Christoph ; Harreither, Wolfgang LU ; Schenkenfelder, Josef ; Schulz, Christopher LU ; Felice, Alfons K.G. ; Gorton, Lo LU and Ludwig, Roland (2023) In Protein Science 32(8).
Abstract

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... (More)

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 sāˆ’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.

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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cytochrome, electron transfer, enzyme engineering, protein interface, surface charge
in
Protein Science
volume
32
issue
8
article number
e4702
publisher
The Protein Society
external identifiers
  • pmid:37312580
  • scopus:85165439831
ISSN
0961-8368
DOI
10.1002/pro.4702
language
English
LU publication?
yes
id
cc115ebd-19ca-4408-96f9-3d345d3e5363
date added to LUP
2023-09-01 13:58:26
date last changed
2024-04-20 02:23:37
@article{cc115ebd-19ca-4408-96f9-3d345d3e5363,
  abstract     = {{<p>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<sup>āˆ’1</sup>. 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.</p>}},
  author       = {{Reichhart, Thomas M.B. and Scheiblbrandner, Stefan and Sygmund, Christoph and Harreither, Wolfgang and Schenkenfelder, Josef and Schulz, Christopher and Felice, Alfons K.G. and Gorton, Lo and Ludwig, Roland}},
  issn         = {{0961-8368}},
  keywords     = {{cytochrome; electron transfer; enzyme engineering; protein interface; surface charge}},
  language     = {{eng}},
  number       = {{8}},
  publisher    = {{The Protein Society}},
  series       = {{Protein Science}},
  title        = {{Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer}},
  url          = {{http://dx.doi.org/10.1002/pro.4702}},
  doi          = {{10.1002/pro.4702}},
  volume       = {{32}},
  year         = {{2023}},
}