Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A

Riin Kont, Jeppe Kari, Kim Borch, Peter Westh, Priit Väljamäe

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of catalytic domain (CD) and a smaller carbohydrate binding module (CBM) connected through the glycosylated linker peptide. A tunnel shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38 in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by complex multi-step mechanism of processive GHs. Here we studied the role of CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind291
Sider (fra-til)26013-26023
ISSN0021-9258
DOI
StatusUdgivet - 2016

Citer dette

@article{1936312757074b29981143d03ec408c2,
title = "Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A",
abstract = "Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of catalytic domain (CD) and a smaller carbohydrate binding module (CBM) connected through the glycosylated linker peptide. A tunnel shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38 in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by complex multi-step mechanism of processive GHs. Here we studied the role of CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.",
author = "Riin Kont and Jeppe Kari and Kim Borch and Peter Westh and Priit V{\"a}ljam{\"a}e",
year = "2016",
doi = "10.1074/jbc.M116.756007",
language = "English",
volume = "291",
pages = "26013--26023",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

}

Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A. / Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit.

I: Journal of Biological Chemistry, Bind 291, 2016, s. 26013-26023.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A

AU - Kont, Riin

AU - Kari, Jeppe

AU - Borch, Kim

AU - Westh, Peter

AU - Väljamäe, Priit

PY - 2016

Y1 - 2016

N2 - Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of catalytic domain (CD) and a smaller carbohydrate binding module (CBM) connected through the glycosylated linker peptide. A tunnel shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38 in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by complex multi-step mechanism of processive GHs. Here we studied the role of CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.

AB - Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of catalytic domain (CD) and a smaller carbohydrate binding module (CBM) connected through the glycosylated linker peptide. A tunnel shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38 in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by complex multi-step mechanism of processive GHs. Here we studied the role of CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.

U2 - 10.1074/jbc.M116.756007

DO - 10.1074/jbc.M116.756007

M3 - Journal article

VL - 291

SP - 26013

EP - 26023

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

ER -