Expression, Purification, and Characterization of a Cobalt-Activated Chitin Deacetylase (Cda2p) from Saccharomyces cerevisiae

Martinou, Aggeliki; Koutsioulis, Dimitris; Bouriotis, Vassilis

doi:10.1006/prep.2001.1547

Cited by 56 publications

(54 citation statements)

References 19 publications

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“…15) Effects of pH and temperature on CDA activity and stability. To determine the optimum pH, the enzyme activity was measured at pH 2-11 at 50 C for 10 min using (GlcNAc) 6 as substrate. The pH stability of CDA was determined by pre-incubating the enzyme in buffer solutions of various pH values for 24 h at 30 C. Residual enzyme activity was determined with the substrate of WSCT-50 at 50 C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…6) Among these, a few CDAs have been confirmed to be glycoprotein, with carbohydrate contents of 18-67% by weight. 3,4,6) Previously, by a liquid medium screening method, we screened an extracellular CDA-producing fungus which was identified to be Mortierella sp. DY-52.…”

Section: A Highly N-glycosylated Chitin Deacetylase Derived From a Nomentioning

confidence: 99%

“…3) As of now, the glycosylation types of all CDAs reported as glycoproteins are N-linked, but not O-linked. For instance, Cda2p from S. cerevisiae 6) and CDA from C. lindemuthianum DSM 63144 4) are N-linked glycoproteins with carbohydrate contents of approximately 18% and 67% by weight, respectively, and a recombinant CDA from Rhizopus circinans 16) consists of 33% N-linked carbohydrate by weight. Here for the first time we describe a highly N-glycosylated CDA from Mortierella sp.…”

Section: Enzymatic Deglycosylationmentioning

confidence: 99%

“…4B), a little higher than the other CDAs (50 C) reported to date. 3,6) The enzyme was stable at temperatures lower than 40 C, and lost 30% of its activity when incubated at 50 C for 2 h.…”

Section: Cda Activity Characterizationmentioning

confidence: 99%

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A HighlyN-Glycosylated Chitin Deacetylase Derived from a Novel Strain ofMortierellasp. DY-52

Zhao

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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Section: Methodsmentioning

confidence: 99%

Section: A Highly N-glycosylated Chitin Deacetylase Derived From a Nomentioning

confidence: 99%

Section: Enzymatic Deglycosylationmentioning

confidence: 99%

“…4B), a little higher than the other CDAs (50 C) reported to date. 3,6) The enzyme was stable at temperatures lower than 40 C, and lost 30% of its activity when incubated at 50 C for 2 h.…”

Section: Cda Activity Characterizationmentioning

confidence: 99%

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A HighlyN-Glycosylated Chitin Deacetylase Derived from a Novel Strain ofMortierellasp. DY-52

Zhao

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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“…ClCDA is a representative member of the CE-4 family and shares a number of sequence motifs, covering several conserved histidine and aspartic acid residues ( Figure 1B). It appears from several kinetic studies that divalent cations may play a role in catalysis (12,19,28,29). Previous studies have shown that ClCDA is an endo-chitin deacetylase with four significant subsites, -2,-1, 0, and +1 (where 0 is the catalytic site), and that subsites -2 and 0 strongly recognize the N-acetyl groups on the GlcNAc sugars whereas subsite +1 recognizes a glucosamine moiety (30)(31)(32).…”

mentioning

confidence: 99%

Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum^,

et al. 2006

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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-HisAsp 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 0 and +1 subsites by (GlcNAc) 2 for activity and proceeds through a tetrahedral oxyanion intermediate.Colletotrichum lindemuthianum is a plant fungal pathogen found extensively in tropical and subtropical regions. Colletotrichum species are the causative agent of anthracnose that affects economically important crop species (1). Furthermore, Colletotrichum sp. have recently been reported to cause subcutaneous and systemic infections among immunosuppressed patients (2). Colletotrichum sp. are facultative biotrophs. Before the fungal hyphae can successfully penetrate and gain access to host tissue, the fungus has to first evade plant antimicrobial hydrolases such as chitinases and -(1,3)glucanases (3, 4). The chitinases degrade fungal chitin, an insoluble linear polymer of -(1-4)-linked N-acetylglucosamine (GlcNAc). 1 The breakdown products may act as elicitors of active defense responses within the plant (5-7). Studies of cell wall composition of invasive fungal hyphae suggest that exposed fungal chitin polymers are partially de-N-acetylated during infection and initial growth within the host (8). Chitosan, the de-N-acetylated product, is a poor substrate for chitinases, which require the presence of N-acetyl moieties for recognition and catalysis (9). Thus, conversion of chitin to chitosan during plant extracellular colonization may protect pathogenic fungal hyphae from being lysed by secreted plant chitinases. The enzyme responsible for chitin modification is a developmentally regulated, secreted, chitin deacetylase (CDA) (10). C. lindemuthianum chitin deacetylase (ClCDA) is a member of the family 4 carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/∼cazy/CAZY (11)], which include several members that share the primary structure assigned as the "NodB homology domain" (12). Rh...

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Identification of a novel carbohydrate esterase fromBjerkandera adusta: Structural and function predictions through bioinformatics analysis and molecular modeling

et al. 2015

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A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg(-1) protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin.

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Expression, Purification, and Characterization of a Cobalt-Activated Chitin Deacetylase (Cda2p) from Saccharomyces cerevisiae

Cited by 56 publications

References 19 publications

A HighlyN-Glycosylated Chitin Deacetylase Derived from a Novel Strain ofMortierellasp. DY-52

A HighlyN-Glycosylated Chitin Deacetylase Derived from a Novel Strain ofMortierellasp. DY-52

Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum^,

Identification of a novel carbohydrate esterase fromBjerkandera adusta: Structural and function predictions through bioinformatics analysis and molecular modeling

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Expression, Purification, and Characterization of a Cobalt-Activated Chitin Deacetylase (Cda2p) from Saccharomyces cerevisiae

Cited by 56 publications

References 19 publications

A HighlyN-Glycosylated Chitin Deacetylase Derived from a Novel Strain ofMortierellasp. DY-52

A HighlyN-Glycosylated Chitin Deacetylase Derived from a Novel Strain ofMortierellasp. DY-52

Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum,

Identification of a novel carbohydrate esterase fromBjerkandera adusta: Structural and function predictions through bioinformatics analysis and molecular modeling

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Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum^,