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

Estefania O Juárez-Hernández,Luz E Casados-Vázquez,Alfredo Torres-Larios,José E Barboza-Corona,Pedro Jimenez-Sandoval,Luis G Brieba

doi:10.1038/s41598-019-39464-z

Abstract

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).

Highlights

IntroductionChitinases are secreted by many prokaryotes and eukaryotes and are utilized for hydrolysis of chitin (a homopolymer of N-acetyl-D-glucosamine, GlcNAc), a rich ubiquitous nutrient resource[1]
Chitinases are secreted by many prokaryotes and eukaryotes and are utilized for hydrolysis of chitin, a rich ubiquitous nutrient resource[1]
B. thuringiensis is most known for the plethora of Cry and Cyt proteins it produces, strains of this microbe produces other proteins that have applied biotechnological value, including chitinases, vegetative insecticidal proteins (Vip) and enhancing-like proteins, antimicrobial peptides and parasporins which can be used to synergize the activity of Cry proteins, to control lepidopteran and coleopteran pests, to inhibit food-borne pathogenic bacteria and to kill human cancer cells of various origins, respectively[1,14,16]

Summary

Introduction

Chitinases are secreted by many prokaryotes and eukaryotes and are utilized for hydrolysis of chitin (a homopolymer of N-acetyl-D-glucosamine, GlcNAc), a rich ubiquitous nutrient resource[1]. Kenyae, has been shown to synergize the insecticidal activity of Cry proteins, and to generate chitin-oligosaccharides with antimicrobial activity[17,18,19] This chitinase has a molecular mass of ~74 kDa, and its bioinformatic analysis predicts that it is composed of four domains: TIM-barrel, chitinase insertion (CID), fibronectin type III (FnIII) and chitin binding domains (CBD)[12]. Our data demonstrate that this enzyme has a modular arrangement formed by four domains: (i) the catalytic region with an (α/β)8-TIM-barrel as a core structure, (ii) the chitinase insertion domain as a barrel insertion, (iii) the fibronectin type III and (iv) a chitin binding domain

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Conclusion