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Interface engineeri...
Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer
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- Reichhart, Thomas M.B. (author)
- University of Natural Resources and Life Sciences, Vienna
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- Scheiblbrandner, Stefan (author)
- University of Natural Resources and Life Sciences, Vienna
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- Sygmund, Christoph (author)
- DirectSens GmbH
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- Harreither, Wolfgang (author)
- University of Natural Resources and Life Sciences, Vienna
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- Schenkenfelder, Josef (author)
- University of Natural Resources and Life Sciences, Vienna
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- Schulz, Christopher (author)
- DirectSens GmbH
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- Felice, Alfons K.G. (author)
- University of Natural Resources and Life Sciences, Vienna
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- Gorton, Lo (author)
- Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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- Ludwig, Roland (author)
- University of Natural Resources and Life Sciences, Vienna
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(creator_code:org_t)
- 2023
- 2023
- English.
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In: Protein Science. - 0961-8368. ; 32:8
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Abstract
Subject headings
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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.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Biokatalys och enzymteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Biocatalysis and Enzyme Technology (hsv//eng)
- NATURVETENSKAP -- Kemi -- Organisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Organic Chemistry (hsv//eng)
Keyword
- cytochrome
- electron transfer
- enzyme engineering
- protein interface
- surface charge
Publication and Content Type
- art (subject category)
- ref (subject category)
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