Journal article
Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases
Journal of the American Chemical Society, Vol.141(36), pp.14451-14459
09/11/2019
Handle:
https://hdl.handle.net/2376/117259
PMID: 31432675
Abstract
Searching for viable strategies to accelerate the catalytic cycle of glycoside hydrolase family 7 (GH7) cellobiohydrolase I (CBHI)-the workhorse cellulose-degrading enzymes, we have performed a total of 12-μs molecular dynamics simulations on GH7 CBHI, which brought to light a new mechanism for cellobiose expulsion, coined "claw-arm" action. The loop flanking the product binding site plays the role of a flexible "arm" extending toward cellobiose, and residue Thr389 of this loop acts as a "claw" that captures cellobiose. Five mutations of residue Thr389 were considered to enhance the loop-cellobiose interaction. The lysine mutant was found to significantly accelerate cellobiose expulsion and facilitate polysaccharide-chain translocation. Lysine mutation of Thr393 in
CBHI (
Cel7A) performed similarly. Lysine approaches the catalytic area and stabilizes the Michaelis complex, potentially affecting glycosylation, the rate-limiting step of the catalytic cycle. QM/MM calculations indicate that lysine replacement diminishes the barrier against proton transfer, the crucial step of glycosylation, by 2.3 kcal/mol. Experimental validation was performed using the full-length wild-type (WT) of
Cel7A and its mutants, recombinantly expressed in
, to degrade the substrates. Compared with the WT, the lysine mutant revealed an associated higher enzymatic reaction rate. Furthermore, cellobiose yield was also increased by lysine mutation, indicating that dissociation of the enzyme from cellulose was accelerated, which largely stems from the enhanced flexibility of the "arm". The present work is envisioned to help design strategies for improving enzymatic activity, while decreasing enzyme cost.
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Details
- Title
- Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases
- Creators
- Zhiyou Zong - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry , Nankai University , Tianjin 300071 , P. R. ChinaQiyu Li - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry , Nankai University , Tianjin 300071 , P. R. ChinaZhangyong Hong - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry , Nankai University , Tianjin 300071 , P. R. ChinaHaohao Fu - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry , Nankai University , Tianjin 300071 , P. R. ChinaWensheng Cai - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry , Nankai University , Tianjin 300071 , P. R. ChinaChristophe Chipot - Department of Physics , University of Illinois at Urbana-Champaign , Urbana , Illinois 61801 , United StatesHuifeng Jiang - Key Laboratory of Systems Microbial Biotechnology, Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering , Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences , Tianjin 300308 , P. R. ChinaDongyuan Zhang - Key Laboratory of Systems Microbial Biotechnology, Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering , Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences , Tianjin 300308 , P. R. ChinaShulin Chen - Key Laboratory of Systems Microbial Biotechnology, Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering , Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences , Tianjin 300308 , P. R. ChinaXueguang Shao - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry , Nankai University , Tianjin 300071 , P. R. China
- Publication Details
- Journal of the American Chemical Society, Vol.141(36), pp.14451-14459
- Academic Unit
- Biological Systems Engineering, Department of
- Identifiers
- 99900582331401842
- Language
- English
- Resource Type
- Journal article