Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites −4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 μM whereas no binding of chitinbiose, -triose or 3′-sialyl-N-acetyllactosamine (Neu5Acα-3Galβ-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (Kd = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites −2 and −3 and particularly the stacking interactions by tryptophanes at subsites −3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNβ-4(GlcNAc)4 by providing a counter charge through substitution of residue T133 at subsite −3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galβ-4(GlcNAc)4.
Original languageEnglish
Pages (from-to)1116 - 1126
JournalGlycobiology
Volume19
Issue number10
DOIs
Publication statusPublished - 13 Jul 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

Chitinases
Trichoderma
Hydrolases
Substrate Specificity
allosamidin
Carbohydrates
Oligosaccharides
Amino Acids
Mutation
Substrates
Trioses
Rubiaceae
Surface Plasmon Resonance
Glycoside Hydrolases
Asparagine
Surface plasmon resonance
Hydrogen Bonding
Glutamine
Aspartic Acid
Glutamic Acid

Keywords

  • protein-carbohydrate interaction
  • surface plasmon resonance
  • chitinase
  • mutagenesis
  • Trichoderma harzianum

Cite this

@article{9d8c67e100ef409bbac132351beb2d03,
title = "Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum",
abstract = "Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites −4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 μM whereas no binding of chitinbiose, -triose or 3′-sialyl-N-acetyllactosamine (Neu5Acα-3Galβ-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (Kd = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites −2 and −3 and particularly the stacking interactions by tryptophanes at subsites −3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNβ-4(GlcNAc)4 by providing a counter charge through substitution of residue T133 at subsite −3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galβ-4(GlcNAc)4.",
keywords = "protein-carbohydrate interaction, surface plasmon resonance, chitinase, mutagenesis, Trichoderma harzianum",
author = "Michael Lienemann and Harry Boer and Arja Paananen and Sylvain Cottaz and Anu Koivula",
year = "2009",
month = "7",
day = "13",
doi = "10.1093/glycob/cwp102",
language = "English",
volume = "19",
pages = "1116 -- 1126",
journal = "Glycobiology",
issn = "0959-6658",
publisher = "Oxford University Press",
number = "10",

}

Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum. / Lienemann, Michael; Boer, Harry; Paananen, Arja; Cottaz, Sylvain; Koivula, Anu.

In: Glycobiology, Vol. 19, No. 10, 13.07.2009, p. 1116 - 1126.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Toward understanding of carbohydrate binding and substrate specificity of a glycosyl hydrolase 18 family (GH-18) chitinase from Trichoderma harzianum

AU - Lienemann, Michael

AU - Boer, Harry

AU - Paananen, Arja

AU - Cottaz, Sylvain

AU - Koivula, Anu

PY - 2009/7/13

Y1 - 2009/7/13

N2 - Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites −4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 μM whereas no binding of chitinbiose, -triose or 3′-sialyl-N-acetyllactosamine (Neu5Acα-3Galβ-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (Kd = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites −2 and −3 and particularly the stacking interactions by tryptophanes at subsites −3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNβ-4(GlcNAc)4 by providing a counter charge through substitution of residue T133 at subsite −3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galβ-4(GlcNAc)4.

AB - Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites −4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 μM whereas no binding of chitinbiose, -triose or 3′-sialyl-N-acetyllactosamine (Neu5Acα-3Galβ-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (Kd = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites −2 and −3 and particularly the stacking interactions by tryptophanes at subsites −3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNβ-4(GlcNAc)4 by providing a counter charge through substitution of residue T133 at subsite −3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galβ-4(GlcNAc)4.

KW - protein-carbohydrate interaction

KW - surface plasmon resonance

KW - chitinase

KW - mutagenesis

KW - Trichoderma harzianum

U2 - 10.1093/glycob/cwp102

DO - 10.1093/glycob/cwp102

M3 - Article

VL - 19

SP - 1116

EP - 1126

JO - Glycobiology

JF - Glycobiology

SN - 0959-6658

IS - 10

ER -