title: Role of the C-terminal and chitin insertion domains on enzymatic activity of endochitinase ChiA74 of Bacillus thuringiensis.
abstract: ChiA74 has modular structure that includes a secretion signal peptide (sp) sequence, and catalytic (CD), chitin insertion (CID), fibronectin type-III (FnIII) and chitin binding (CBD) domains. We described for the first time the existence of a putative CID in ChiA74. Mature ChiA74 lacking its sp sequence (rChiA74Δsp, ∼70kDa) and two truncated versions, rChiA74Δsp-60, rChiA74Δsp-50 lacking, respectively, CBD and CDB-FnIII were produced. rChiA74Δsp and rChiA74Δsp-60 are unstable and were processed to generate stable proteins of ∼50kDa. With colloidal chitin, rChiA74Δsp and rChiA74Δsp-50 had higher activity than rChiA74Δsp-60. rChiA74Δsp showed similar ability to bind chitin than rChiA74Δsp-50. The catalytic efficiencies (kcat/Km) of rChiA74Δsp and rChiA74Δsp-50 were higher, ∼ 21-fold than rChiA74Δsp-60, using chitin as the substrate. Optimal activity was detected at pH 7 and 40°C. Data suggest that the CBD in ChiA74 is important for binding to chitin, but not necessary as the presence of a CID together with the CD in a stable truncated version (i.e. ChiA74Δsp-50) has similar affinity and hydrolytic activity as the mature enzyme. The CID of ChiA74 showed identities of ∼ 55% with CIDs of other chitinases such as those from B. circulans and B. licheniformis, respectively, and conserved residues important for interacting with chitin.
authors.affiliation: University of Guanajuato Campus Irapuato-Salamanca, Life Science Division, Graduate Program in Biosciences, 36500, México.
authors.forename: Estefania O
authors.initials: EO
authors.lastname: Juárez-Hernández
authors.affiliation: University of Guanajuato Campus Irapuato-Salamanca, Life Science Division, Graduate Program in Biosciences, 36500, México; Department of Food, University of Guanajuato Campus Irapuato-Salamanca, Life Science Division, 36500, México.
authors.forename: Luz E
authors.initials: LE
authors.lastname: Casados-Vázquez
authors.affiliation: California Baptist University, Department of Biological Sciences, 8432 Magnolia Avenue, Riverside, CA 92504, United States; Department of Entomology, University of California, Riverside, Riverside, CA 92521, United States.
authors.forename: Dennis K
authors.initials: DK
authors.lastname: Bideshi
authors.affiliation: University of Guanajuato Campus Irapuato-Salamanca, Life Science Division, Graduate Program in Biosciences, 36500, México; Department of Food, University of Guanajuato Campus Irapuato-Salamanca, Life Science Division, 36500, México.
authors.forename: R
authors.initials: R
authors.lastname: Salcedo-Hernández
authors.affiliation: University of Guanajuato Campus Irapuato-Salamanca, Life Science Division, Graduate Program in Biosciences, 36500, México; Department of Food, University of Guanajuato Campus Irapuato-Salamanca, Life Science Division, 36500, México. Electronic address: josebar@ugto.mx.
authors.forename: José E
authors.initials: JE
authors.lastname: Barboza-Corona
mesh_terms: D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000201:enzymology*; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000494:pharmacology; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D002686:Chitin; D002688:Chitinases
keywords: Bacillus thuringiensis; ChiA74; Chitin insertion domain