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Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes.

Viens P, Lacombe-Harvey MÈ, Brzezinski R - Mar Drugs (2015)

Bottom Line: Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan.Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level.This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

View Article: PubMed Central - PubMed

Affiliation: Biologie, Faculté des Sciences, Université de Sherbrooke, 2500, boul. de l'Université, Sherbrooke, QC J1K 2R1, Canada. Pascal.Viens@USherbrooke.ca.

ABSTRACT
Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

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Tertiary structure of chitosanase from Microbacterium sp. OU01: distribution of substrate-binding residues among six subsites [33,53]. The colors assigned to the various subsites are shown in the upper right part of the figure. Residues painted with two colors participate simultaneously to two subsites.
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marinedrugs-13-06566-f005: Tertiary structure of chitosanase from Microbacterium sp. OU01: distribution of substrate-binding residues among six subsites [33,53]. The colors assigned to the various subsites are shown in the upper right part of the figure. Residues painted with two colors participate simultaneously to two subsites.

Mentions: In early structural studies, the enzyme residues potentially interacting with the chitosan substrate were identified by modeling of the mode of binding of chitosan oligosaccharides (mainly hexamers of d-glucosamine) with chitosanase. A first model build for the Streptomyces sp. N174 chitosanase and based on the mode of action of lysozyme [32] suggested the presence of six subsites, named A to F, with an asymmetrical cleavage of “4 + 2” type occurring between subsites D and E. Accordingly, the hydrolysis of (GlcN)6 should yield dimeric and tetrameric products in equimolar proportions. However, kinetic data showed that the symmetrical “3 + 3”-type splitting is much favored over the asymmetrical one [60]. The symmetrical model was confirmed recently when Lyu et al. obtained a crystal of the chitosanase OU01 mutated at the general acid residue, complexed with the hexaglucosamine substrate [33]. The authors provided, for the first time, a description of the substrate-binding mechanism based on direct crystallographic observations. The substrate-interacting residues are shown in Figure 5. They are also listed in Table 1, together with the corresponding residues from other chitosanases which structures have been elucidated. Lyu et al. (2014) emphasize again the importance of acidic residues in the substrate binding cleft [33]. The −2 subsite is one of the most important determinants of the specificity of OU01 enzyme as a chitosanase, where the substrate interacts with two highly conserved residues: Arg45 and Asp60. This observation confirmed previous studies by site-directed mutagenesis, which showed that mutations of corresponding residues in other chitosanases resulted in severe impairment of enzymatic activity [49,55,57]. Performing a series of mutations of residues in the substrate-binding cleft, Lyu et al. concluded that, “the subsites −2, −1 and +1 are probably the dominant contributors for substrate binding and essential for hydrolysis” [33].


Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes.

Viens P, Lacombe-Harvey MÈ, Brzezinski R - Mar Drugs (2015)

Tertiary structure of chitosanase from Microbacterium sp. OU01: distribution of substrate-binding residues among six subsites [33,53]. The colors assigned to the various subsites are shown in the upper right part of the figure. Residues painted with two colors participate simultaneously to two subsites.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663542&req=5

marinedrugs-13-06566-f005: Tertiary structure of chitosanase from Microbacterium sp. OU01: distribution of substrate-binding residues among six subsites [33,53]. The colors assigned to the various subsites are shown in the upper right part of the figure. Residues painted with two colors participate simultaneously to two subsites.
Mentions: In early structural studies, the enzyme residues potentially interacting with the chitosan substrate were identified by modeling of the mode of binding of chitosan oligosaccharides (mainly hexamers of d-glucosamine) with chitosanase. A first model build for the Streptomyces sp. N174 chitosanase and based on the mode of action of lysozyme [32] suggested the presence of six subsites, named A to F, with an asymmetrical cleavage of “4 + 2” type occurring between subsites D and E. Accordingly, the hydrolysis of (GlcN)6 should yield dimeric and tetrameric products in equimolar proportions. However, kinetic data showed that the symmetrical “3 + 3”-type splitting is much favored over the asymmetrical one [60]. The symmetrical model was confirmed recently when Lyu et al. obtained a crystal of the chitosanase OU01 mutated at the general acid residue, complexed with the hexaglucosamine substrate [33]. The authors provided, for the first time, a description of the substrate-binding mechanism based on direct crystallographic observations. The substrate-interacting residues are shown in Figure 5. They are also listed in Table 1, together with the corresponding residues from other chitosanases which structures have been elucidated. Lyu et al. (2014) emphasize again the importance of acidic residues in the substrate binding cleft [33]. The −2 subsite is one of the most important determinants of the specificity of OU01 enzyme as a chitosanase, where the substrate interacts with two highly conserved residues: Arg45 and Asp60. This observation confirmed previous studies by site-directed mutagenesis, which showed that mutations of corresponding residues in other chitosanases resulted in severe impairment of enzymatic activity [49,55,57]. Performing a series of mutations of residues in the substrate-binding cleft, Lyu et al. concluded that, “the subsites −2, −1 and +1 are probably the dominant contributors for substrate binding and essential for hydrolysis” [33].

Bottom Line: Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan.Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level.This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

View Article: PubMed Central - PubMed

Affiliation: Biologie, Faculté des Sciences, Université de Sherbrooke, 2500, boul. de l'Université, Sherbrooke, QC J1K 2R1, Canada. Pascal.Viens@USherbrooke.ca.

ABSTRACT
Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

Show MeSH