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Characterization of a Bacillus thuringiensis chitinase that binds to cellulose and chitin.

ABSTRACT: Bacillus thuringiensis is a Gram-positive soil bacterium that is known to be a bacterial biopesticide that produces insecticidal proteins called crystal proteins (Cry). In the insecticidal process, chitinases are suggested to perforate the peritrophic membrane barrier to facilitate the invasion of the Cry proteins into epithelial membranes. A chitinase gene from B. thuringiensis was successfully expressed in a soluble form in Escherichia coli, and the gene product was purified and characterized. The purified recombinant enzyme, BthChi74, hydrolyzed an artificial substrate, 4-nitrophenyl N,N'-diacetyl-?-D-chitobioside [4NP-(GlcNAc)2], and the natural substrates, colloidal chitin and crystalline ?-chitin, but it did not hydrolyze cellulose. BthChi74 exhibited catalytic activity under a weakly acidic to neutral pH range at 50 °C, and it was stable over a wide pH range for 24 h. Differential scanning fluorimetry (DSF) indicated a protein melting temperature (T m) of 63.6 °C. Kinetic analysis revealed k cat and K M values of 1.5 s-1 and 159 ?M, respectively, with 4NP-(GlcNAc)2 as a substrate. BthChi74 produced (GlcNAc)2 and GlcNAc from colloidal chitin and ?-chitin as substrates, but the activity toward the latter was lower than that toward the former. BthChi74 could bind similarly to chitin beads, crystalline ?-chitin, and cellulose through a unique family 2 carbohydrate-binding module (CBM2). The structure-function relationships of BthChi74 are discussed in relation to other chitinases, such as Listeria chitinase, which possesses a family 5 carbohydrate-binding module (CBM5).

SUBMITTER: Honda S

PROVIDER: S-EPMC5328894 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Characterization of a Bacillus thuringiensis chitinase that binds to cellulose and chitin.

Honda Shotaro S Kunii Toshiyuki T Nohara Kenta K Wakita Satoshi S Sugahara Yasusato Y Kawakita Masao M Oyama Fumitaka F Sakaguchi Masayoshi M

AMB Express 20170228 1

Bacillus thuringiensis is a Gram-positive soil bacterium that is known to be a bacterial biopesticide that produces insecticidal proteins called crystal proteins (Cry). In the insecticidal process, chitinases are suggested to perforate the peritrophic membrane barrier to facilitate the invasion of the Cry proteins into epithelial membranes. A chitinase gene from B. thuringiensis was successfully expressed in a soluble form in Escherichia coli, and the gene product was purified and characterized. ...[more]

PMID: 28244030

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Project description:Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)(n), into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67-Thr-69 and Ile-106-Val-112) to interact with (NAG)(n), generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ?4.6 Å along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)(n) reveal (NAG)(2), (NAG)(2), and (NAG)(4) in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)(4); in an intermediate state, E145Q/Y227F with a boat-form NAG at the -1 subsite, -1-(NAG); after hydrolysis, E145Q with a chair form -1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between -1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of -1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of -1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)(n) provide new insights into its substrate binding and the mechanistic action.

Dataset Information

Characterization of a Bacillus thuringiensis chitinase that binds to cellulose and chitin.

Publications

Characterization of a Bacillus thuringiensis chitinase that binds to cellulose and chitin.

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