Abstract
A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)4-6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)2 and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)2 or GlcNAc, which are easily taken up by the cells.
Highlights
A cDNA of putative chitinase from Euglena gracilis, designated E. gracilis chitinase-A (EgChiA), encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix
The sequence of 23 residues (Leu938 to Cys960) at the C-terminus was predicted to be a transmembrane helix using the programs TMHMM2.0 and SOSUI (Supplementary Figure 2). These analyses suggested that EgChiA is located at the cell surface
EgChiA consists of two GH18 catalytic domains and two CBM18 chitin-binding domains, belonging to the PF-ChiA-like GH18 sub-family and ChBD1_1 family, respectively, linked by proline, serine, and threonine-rich regions
Summary
A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. Chitinases (EC 3.2.1.14) catalyze the hydrolysis of chitin, which is a β-1,4-linked homopolymer or an oligomer of N-acetylglucosamine (GlcNAc) These enzymes have been divided in two families: glycoside hydrolase family (GH18) and (GH19), based on the amino acid sequences of their catalytic modules, according to the CAZy database (http://www.cazy.org/) [1]. One of the unique features of EgChiA was the transmembrane domain found at the C-terminus, in addition to two catalytic domains and BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY two chitin-binding domains This type of domain organization was identified for the first time for chitinases in Excavata. One of the physiological roles of plant and animal chitinases is to protect the host against fungal pathogens by degrading chitin, a major component of the cell wall of many fungi [7,8]. We will discuss the physiological role of EgChiA based on our structural and functional data
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