Structure and mechanism of chitin deacetylase from the fungal pathogen colletotrichum lindemuthianum

David E. Blair, Omid Hekmat, Alexander W. Schüttelkopf, Binesh Shrestha, Ken Tokuyasu, Stephen G. Withers, Daan M. F. van Aalten

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 Å crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-His-Asp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the O and +1 subsites by (GlcNAc)2 for activity and proceeds through a tetrahedral oxyanion intermediate.

Original languageEnglish
Pages (from-to)9416-9426
Number of pages11
JournalBiochemistry
Volume45
Issue number31
DOIs
Publication statusPublished - 8 Aug 2006

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chitin deacetylase
Colletotrichum
Chitin
Pathogens
Acetylesterase
His-His-His-His-His-His
Atomic spectroscopy
Acids
Substrates
Substrate Specificity
Catalysis
Absorption spectroscopy
Histidine
Aspartic Acid
Free energy
Purification
Zinc
Catalytic Domain
Spectrum Analysis
Crystal structure

Cite this

Blair, D. E., Hekmat, O., Schüttelkopf, A. W., Shrestha, B., Tokuyasu, K., Withers, S. G., & van Aalten, D. M. F. (2006). Structure and mechanism of chitin deacetylase from the fungal pathogen colletotrichum lindemuthianum. Biochemistry, 45(31), 9416-9426. https://doi.org/10.1021/bi0606694
Blair, David E. ; Hekmat, Omid ; Schüttelkopf, Alexander W. ; Shrestha, Binesh ; Tokuyasu, Ken ; Withers, Stephen G. ; van Aalten, Daan M. F. / Structure and mechanism of chitin deacetylase from the fungal pathogen colletotrichum lindemuthianum. In: Biochemistry. 2006 ; Vol. 45, No. 31. pp. 9416-9426.
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Blair, DE, Hekmat, O, Schüttelkopf, AW, Shrestha, B, Tokuyasu, K, Withers, SG & van Aalten, DMF 2006, 'Structure and mechanism of chitin deacetylase from the fungal pathogen colletotrichum lindemuthianum', Biochemistry, vol. 45, no. 31, pp. 9416-9426. https://doi.org/10.1021/bi0606694

Structure and mechanism of chitin deacetylase from the fungal pathogen colletotrichum lindemuthianum. / Blair, David E.; Hekmat, Omid; Schüttelkopf, Alexander W.; Shrestha, Binesh; Tokuyasu, Ken; Withers, Stephen G.; van Aalten, Daan M. F.

In: Biochemistry, Vol. 45, No. 31, 08.08.2006, p. 9416-9426.

Research output: Contribution to journalArticle

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AU - Blair, David E.

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AU - Schüttelkopf, Alexander W.

AU - Shrestha, Binesh

AU - Tokuyasu, Ken

AU - Withers, Stephen G.

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N2 - The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 Å crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-His-Asp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the O and +1 subsites by (GlcNAc)2 for activity and proceeds through a tetrahedral oxyanion intermediate.

AB - The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 Å crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-His-Asp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the O and +1 subsites by (GlcNAc)2 for activity and proceeds through a tetrahedral oxyanion intermediate.

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Blair DE, Hekmat O, Schüttelkopf AW, Shrestha B, Tokuyasu K, Withers SG et al. Structure and mechanism of chitin deacetylase from the fungal pathogen colletotrichum lindemuthianum. Biochemistry. 2006 Aug 8;45(31):9416-9426. https://doi.org/10.1021/bi0606694