Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello‐oligomers show high flexibility in the substrate binding

Tarja Parkkinen, Anu Koivula, Jari Vehmaanperä, Juha Rouvinen (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

37 Citations (Scopus)

Abstract

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single‐module, cellulose‐degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello‐oligomers: cellobiose (Glc2), cellotriose (Glc3), and cellotetraose (Glc4), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β‐sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello‐oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello‐oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (4C1) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.
Original languageEnglish
Pages (from-to)1383 - 1394
Number of pages12
JournalProtein Science
Volume17
Issue number8
DOIs
Publication statusPublished - 2008
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Crystal structure
Conformations
Tunnels
Substrates
Enzymes
Cellobiose
Sandwich structures
Glycoside Hydrolases
Oligosaccharides
Cellulose
Catalyst activity
Catalytic Domain
Amino Acids
Molecules
Temperature
cellotetraose

Keywords

  • cellulase
  • cellobiohydrolase
  • substrate complex
  • crystal structure
  • reaction mechanism
  • Melanocarpus albomyces
  • thermophilic

Cite this

@article{1d7f553eb2da418ead14bbaea7f81834,
title = "Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello‐oligomers show high flexibility in the substrate binding",
abstract = "Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single‐module, cellulose‐degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello‐oligomers: cellobiose (Glc2), cellotriose (Glc3), and cellotetraose (Glc4), at high resolution (1.6–2.1 {\AA}). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β‐sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello‐oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello‐oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (4C1) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.",
keywords = "cellulase, cellobiohydrolase, substrate complex, crystal structure, reaction mechanism, Melanocarpus albomyces, thermophilic",
author = "Tarja Parkkinen and Anu Koivula and Jari Vehmaanper{\"a} and Juha Rouvinen",
year = "2008",
doi = "10.1110/ps.034488.108",
language = "English",
volume = "17",
pages = "1383 -- 1394",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Wiley-Blackwell",
number = "8",

}

Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello‐oligomers show high flexibility in the substrate binding. / Parkkinen, Tarja; Koivula, Anu; Vehmaanperä, Jari; Rouvinen, Juha (Corresponding Author).

In: Protein Science, Vol. 17, No. 8, 2008, p. 1383 - 1394.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello‐oligomers show high flexibility in the substrate binding

AU - Parkkinen, Tarja

AU - Koivula, Anu

AU - Vehmaanperä, Jari

AU - Rouvinen, Juha

PY - 2008

Y1 - 2008

N2 - Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single‐module, cellulose‐degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello‐oligomers: cellobiose (Glc2), cellotriose (Glc3), and cellotetraose (Glc4), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β‐sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello‐oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello‐oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (4C1) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.

AB - Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single‐module, cellulose‐degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello‐oligomers: cellobiose (Glc2), cellotriose (Glc3), and cellotetraose (Glc4), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β‐sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello‐oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello‐oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (4C1) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.

KW - cellulase

KW - cellobiohydrolase

KW - substrate complex

KW - crystal structure

KW - reaction mechanism

KW - Melanocarpus albomyces

KW - thermophilic

U2 - 10.1110/ps.034488.108

DO - 10.1110/ps.034488.108

M3 - Article

VL - 17

SP - 1383

EP - 1394

JO - Protein Science

JF - Protein Science

SN - 0961-8368

IS - 8

ER -