Abstract
Insect chitin deacetylases (CDAs) catalyze the removal of acetyl groups from chitin and modify this polymer during its synthesis and reorganization. CDAs are essential for insect survival and therefore represent promising targets for insecticide development. However, the structural and biochemical characteristics of insect CDAs have remained elusive. Here, we report the crystal structures of two insect CDAs from the silk moth Bombyx mori: BmCDA1, which may function in cuticle modification, and BmCDA8, which may act in modifying peritrophic membranes in the midgut. Both enzymes belong to the carbohydrate esterase 4 (CE4) family. Comparing their overall structures at 1.98–2.4 Å resolution with those from well-studied microbial CDAs, we found that two unique loop regions in BmCDA1 and BmCDA8 contribute to the distinct architecture of their substrate-binding clefts. These comparisons revealed that both BmCDA1 and BmCDA8 possess a much longer and wider substrate-binding cleft with a very open active site in the center than the microbial CDAs, including VcCDA from Vibrio cholerae and ArCE4A from Arthrobacter species AW19M34-1. Biochemical analyses indicated that BmCDA8 is an active enzyme that requires its substrates to occupy subsites 0, +1, and +2 for catalysis. In contrast, BmCDA1 also required accessory proteins for catalysis. To the best of our knowledge, our work is the first to unveil the structural and biochemical features of insect proteins belonging to the CE4 family.
Highlights
Insect chitin deacetylases (CDAs) catalyze the removal of acetyl groups from chitin and modify this polymer during its synthesis and reorganization
Our experiments showed that full-length recombinant BmCDA1 was not stable and underwent autocleavage when incubated with the crystallization reagent
Because the biochemical data indicated that BmCDA8 was not able to deacetylate GlcN nor monodeacetylated products of (GlcNAc) or (GlcNAc)2 (Table 2), we deduced that BmCDA8 activity requires substrates to occupy subsites 0, ϩ1, and ϩ2 (Fig. 3C), where 0 is the catalytic site, and the plus sign refers to the reducing ends, according to the nomenclature commonly used for carbohydrate esterase 4 (CE4) enzymes (40 –42)
Summary
Insects possess a greater number of CDAs than any other organism. An insect may possess as many as five groups of genes encoding CDAs [32], which may function in the epidermis, tracheal tubes, and midgut [33,34,35,36]. Two cda gene mutants of Drosophila melanogaster embryos resulted in elongated and tortuous tracheal tubes [34, 35]. RNAi of all nine cda genes from Tribolium castaneum resulted in abnormal phenotypes in the tracheal tubes and cuticle and in joint defects, followed by molting failure or even death [33]. Many efforts have failed to demonstrate the deacetylation activities of insect CDAs toward chitinous substrates in vitro [30, 39]. This work will assist the development of specific agrochemicals for pest control
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