The crystal structure of the chitinase ChiA74 of Bacillus thuringiensis has a multidomain assembly.

There is no structural information about any chitinase synthesized by Bacillus thuringiensis, the most successful microbial insect larvicide used worldwide. In this study, we solved the 3D structure of the chitinase ChiA74 at 2.26 Å. The crystal structure shows that ChiA74 is composed of a modular arrangement formed by (i) a catalytic region (CD), (ii) a chitinase insertion domain (CID), (iii) a fibronectin type III domain (FnIII), and (iv) a chitin binding domain (CBD). The location of the CBD with respect to the CD has no structural similarity to other chitinases with known structures. The activity of a ChiA74 lacking its secretion signal peptide (ChiA74Δsp) and a truncated version lacking its CBD/FnIII domains (ChiA74Δsp-50) did not have statistical differences in activity against colloidal chitin. However, ChiA74Δsp exhibits 4.5 and 2.0 higher activity than versions lacking the CBD (ChiA74Δsp-60) and CBD/FnIII domains (ChiA74Δsp-50), respectively, when crystalline chitin was used as substrate. Our data suggest that the CBD might plays a significant role in crystalline chitin hydrolysis. We also demonstrated the importance of the catalytic E211 in the CD, as mutants ChiA74ΔspE211N and ChiA74ΔspD207N, E211N were inactive against colloidal and crystalline chitins, chitosan and 4-MU-GlcNAc3. ChiA74 has a processive activity producing oligosaccharides with degree of polymerization (DP) of 1 (GlcNAc) and 2 (GlcNAc2).

Role of the C-terminal and chitin insertion domains on enzymatic activity of endochitinase ChiA74 of Bacillus thuringiensis.

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.