Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands

Tamás Ponyi, Lóránd Szabó, Tibor Nagy, L. Orosz, Peter J. Simpson, Michael P. Williamson, Harry J. Gilbert

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM1 Os), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N- bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabo, L., Nagy, T., Gilbert, H. J., and Williamson, M.P. (2000) Biochemistry 39, 978-984].

Original languageEnglish
Pages (from-to)985-991
Number of pages7
JournalBiochemistry
Volume39
Issue number5
DOIs
Publication statusPublished - Feb 8 2000

Fingerprint

Endo-1,4-beta Xylanases
Pseudomonas
Cellulose
Ligands
Pseudomonas fluorescens
Bromosuccinimide
Cellulases
Aromatic Amino Acids
Mutation
Biochemistry
Proteins
Glucans
Escherichia coli Proteins
Mutant Proteins
Titration
Hydrophobic and Hydrophilic Interactions
Tryptophan
Sugars
Alanine
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

Cite this

Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands. / Ponyi, Tamás; Szabó, Lóránd; Nagy, Tibor; Orosz, L.; Simpson, Peter J.; Williamson, Michael P.; Gilbert, Harry J.

In: Biochemistry, Vol. 39, No. 5, 08.02.2000, p. 985-991.

Research output: Contribution to journalArticle

Ponyi, Tamás ; Szabó, Lóránd ; Nagy, Tibor ; Orosz, L. ; Simpson, Peter J. ; Williamson, Michael P. ; Gilbert, Harry J. / Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands. In: Biochemistry. 2000 ; Vol. 39, No. 5. pp. 985-991.
@article{eb0f195fb90e4a37892ed937f1773e51,
title = "Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands",
abstract = "Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM1 Os), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N- bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabo, L., Nagy, T., Gilbert, H. J., and Williamson, M.P. (2000) Biochemistry 39, 978-984].",
author = "Tam{\'a}s Ponyi and L{\'o}r{\'a}nd Szab{\'o} and Tibor Nagy and L. Orosz and Simpson, {Peter J.} and Williamson, {Michael P.} and Gilbert, {Harry J.}",
year = "2000",
month = "2",
day = "8",
doi = "10.1021/bi9921642",
language = "English",
volume = "39",
pages = "985--991",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",

}

TY - JOUR

T1 - Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands

AU - Ponyi, Tamás

AU - Szabó, Lóránd

AU - Nagy, Tibor

AU - Orosz, L.

AU - Simpson, Peter J.

AU - Williamson, Michael P.

AU - Gilbert, Harry J.

PY - 2000/2/8

Y1 - 2000/2/8

N2 - Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM1 Os), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N- bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabo, L., Nagy, T., Gilbert, H. J., and Williamson, M.P. (2000) Biochemistry 39, 978-984].

AB - Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM1 Os), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N- bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabo, L., Nagy, T., Gilbert, H. J., and Williamson, M.P. (2000) Biochemistry 39, 978-984].

UR - http://www.scopus.com/inward/record.url?scp=0034620561&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034620561&partnerID=8YFLogxK

U2 - 10.1021/bi9921642

DO - 10.1021/bi9921642

M3 - Article

C2 - 10653642

AN - SCOPUS:0034620561

VL - 39

SP - 985

EP - 991

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -