Abstract
BackgroundVibrio carchariae chitinase A (EC3.2.1.14) is a family-18 glycosyl hydrolase and comprises three distinct structural domains: i) the amino terminal chitin binding domain (ChBD); ii) the (α/β)8 TIM barrel catalytic domain (CatD); and iii) the α + β insertion domain. The predicted tertiary structure of V. carchariae chitinase A has located the residues Ser33 & Trp70 at the end of ChBD and Trp231 & Tyr245 at the exterior of the catalytic cleft. These residues are surface-exposed and presumably play an important role in chitin hydrolysis.ResultsPoint mutations of the target residues of V. carchariae chitinase A were generated by site-directed mutagenesis. With respect to their binding activity towards crystalline α-chitin and colloidal chitin, chitin binding assays demonstrated a considerable decrease for mutants W70A and Y245W, and a notable increase for S33W and W231A. When the specific hydrolyzing activity was determined, mutant W231A displayed reduced hydrolytic activity, whilst Y245W showed enhanced activity. This suggested that an alteration in the hydrolytic activity was not correlated with a change in the ability of the enzyme to bind to chitin polymer. A mutation of Trp70 to Ala caused the most severe loss in both the binding and hydrolytic activities, which suggested that it is essential for crystalline chitin binding and hydrolysis. Mutations varied neither the specific hydrolyzing activity against pNP-[GlcNAc]2, nor the catalytic efficiency against chitohexaose, implying that the mutated residues are not important in oligosaccharide hydrolysis.ConclusionOur data provide direct evidence that the binding as well as hydrolytic activities of V. carchariae chitinase A to insoluble chitin are greatly influenced by Trp70 and less influenced by Ser33. Though Trp231 and Tyr245 are involved in chitin hydrolysis, they do not play a major role in the binding process of crystalline chitin and the guidance of the chitin chain into the substrate binding cleft of the enzyme.
Highlights
Vibrio carchariae chitinase A (EC3.2.1.14) is a family-18 glycosyl hydrolase and comprises three distinct structural domains: i) the amino terminal chitin binding domain (ChBD); ii) the (α/β)8 TIM barrel catalytic domain (CatD); and iii) the α + β insertion domain
In the carbohydrate active enzymes (CAZy) database http://www.cazy.org/, carbohydrate enzymes are first classified as glycosyl hydrolases (GH), glycosyl transferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), and carbohydrate binding modules (CBM), and further divided into numbered families with structurally-related catalytic and carbohydrate-binding modules
We have investigated the functional roles of four amino acid residues (Ser33, Trp70, Trp231 and Tyr245) at the surface of V. carchariae chitinase A
Summary
Vibrio carchariae chitinase A (EC3.2.1.14) is a family-18 glycosyl hydrolase and comprises three distinct structural domains: i) the amino terminal chitin binding domain (ChBD); ii) the (α/β) TIM barrel catalytic domain (CatD); and iii) the α + β insertion domain. Chitin polymer has been presumed to unidirectionally enter the substrate binding cleft of chitinases under the guidance of a few surface-exposed residues at the exterior of the substrate binding cleft [21,22] Those residues were identified as Trp, Trp, Phe232, and Trp245 in S. marcescens Chi A [18], Ser, Trp, Trp232, and Trp245 in Aeromonas caviae Chi1 [23], and Trp122 and Trp134 in B. circulans Chi A1 [17]. Site-directed mutagenesis was employed in combination with following chitin binding assays and kinetic analysis to investigate the significance of the putative surface-exposed residues Ser, Trp, Trp231 and Tyr245 for the binding and hydrolytic activities of the Vibrio chitinase A
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
