Differential recognition of animal type β4-galactosylated and α3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum

Harry Boer (Corresponding Author), Nana Munck, Jari Natunen, Gerd Wohlfahrt, Hans Söderlund, Ossi Renkonen, Anu Koivula

Research output: Contribution to journalArticleScientificpeer-review

27 Citations (Scopus)

Abstract

We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (α/β)8-barrel fold enzymes to recognize β-1,4-galactosylated and α-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, β-1,4-galactosylated and α-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (−2−1–+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites −4 and −3 of Chit42, and at the subsite −4 of Chit33. Fucose units can be accepted as a branch at the putative −3 and −4 sites of Chit33 and as a branch point at −3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.
Original languageEnglish
Pages (from-to)1303 - 1313
Number of pages11
JournalGlycobiology
Volume14
Issue number12
DOIs
Publication statusPublished - 2004
MoE publication typeA1 Journal article-refereed

Fingerprint

Chitinases
Trichoderma
Oligosaccharides
Animals
Substrates
Protein Engineering
Fucose
Structural Models
Hydrolases
Substrate Specificity
Fungi
Catalysis
Galactose
Sugars
Purification
Hydrolysis
Proteins
Binding Sites
Kinetics
Enzymes

Keywords

  • (alfa/beta)8-barrel fold
  • chitinases
  • chito-oligosaccharides
  • molecular modeling
  • substrate specificity

Cite this

Boer, Harry ; Munck, Nana ; Natunen, Jari ; Wohlfahrt, Gerd ; Söderlund, Hans ; Renkonen, Ossi ; Koivula, Anu. / Differential recognition of animal type β4-galactosylated and α3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum. In: Glycobiology. 2004 ; Vol. 14, No. 12. pp. 1303 - 1313.
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title = "Differential recognition of animal type β4-galactosylated and α3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum",
abstract = "We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (α/β)8-barrel fold enzymes to recognize β-1,4-galactosylated and α-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, β-1,4-galactosylated and α-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (−2−1–+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites −4 and −3 of Chit42, and at the subsite −4 of Chit33. Fucose units can be accepted as a branch at the putative −3 and −4 sites of Chit33 and as a branch point at −3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.",
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Differential recognition of animal type β4-galactosylated and α3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum. / Boer, Harry (Corresponding Author); Munck, Nana; Natunen, Jari; Wohlfahrt, Gerd; Söderlund, Hans; Renkonen, Ossi; Koivula, Anu.

In: Glycobiology, Vol. 14, No. 12, 2004, p. 1303 - 1313.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Differential recognition of animal type β4-galactosylated and α3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum

AU - Boer, Harry

AU - Munck, Nana

AU - Natunen, Jari

AU - Wohlfahrt, Gerd

AU - Söderlund, Hans

AU - Renkonen, Ossi

AU - Koivula, Anu

PY - 2004

Y1 - 2004

N2 - We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (α/β)8-barrel fold enzymes to recognize β-1,4-galactosylated and α-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, β-1,4-galactosylated and α-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (−2−1–+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites −4 and −3 of Chit42, and at the subsite −4 of Chit33. Fucose units can be accepted as a branch at the putative −3 and −4 sites of Chit33 and as a branch point at −3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.

AB - We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (α/β)8-barrel fold enzymes to recognize β-1,4-galactosylated and α-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, β-1,4-galactosylated and α-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (−2−1–+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites −4 and −3 of Chit42, and at the subsite −4 of Chit33. Fucose units can be accepted as a branch at the putative −3 and −4 sites of Chit33 and as a branch point at −3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.

KW - (alfa/beta)8-barrel fold

KW - chitinases

KW - chito-oligosaccharides

KW - molecular modeling

KW - substrate specificity

U2 - 10.1093/glycob/cwh121

DO - 10.1093/glycob/cwh121

M3 - Article

VL - 14

SP - 1303

EP - 1313

JO - Glycobiology

JF - Glycobiology

SN - 0959-6658

IS - 12

ER -