Structural and biochemical insights into the catalytic mechanisms of two insect chitin deacetylases of the carbohydrate esterase 4 family

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 : Bm CDA1, which may function in cuticle modification, and Bm CDA8, 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 Bm CDA1 and Bm CDA8 contribute to the distinct architecture of their substrate-binding clefts. These comparisons revealed that both Bm CDA1 and Bm CDA8 possess a much longer and wider substrate-binding cleft with a very open active site in the center than the microbial CDAs, including Vc CDA from Vibrio cholerae and Ar CE4A from Arthrobacter species AW19M34-1. Biochemical analyses indicated that Bm CDA8 is an active enzyme that requires its substrates to occupy subsites 0, +1, and +2 for catalysis. In contrast, Bm CDA1 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.

A novel tick protein supports integrity of gut peritrophic matrix impacting existence of gut microbiome and Lyme disease pathogens

“…Murine skin tissues were collected 12 days after tick repletion, and the B. burgdorferi burden was detected using qPCR. ns, non-significant; *p > .05 modes (Liu et al, 2019). Therefore, PM_CBP, which harbours a CDA- We found that interference with PM_CBP expression reduces the thickness of the Ixodes PM and impacts its integrity, thereby increasing its permeability to large molecules.…”

“…In silico analyses have indicated that PM_CBP has a putative domain that belongs to the carbohydrate esterase 4 (CE4) superfamily, which includes the CDA proteins that catalyse the hydrolysis of acetyl groups from chitin to chitosan (Dixit et al, 2008; Luschnig, Batz, Armbruster, & Krasnow, 2006; Tsigos, Martinou, Kafetzopoulos, & Bouriotis, 2000). The crystal structures of several CDAs are available, including ones from insects, which together imply the conservation of critical residues in the active site (Liu et al, 2019), although extensive variations among these enzymes in the shapes of their substrate‐binding sites could lead to disparate substrate preferences and deacetylation modes (Liu et al, 2019). Therefore, PM_CBP, which harbours a CDA‐like domain, is expected to perform roles in the modification of PM structure and organisation, although its precise function remains unknown.…”

“…The crystal structures of several CDAs are available, including ones from insects, which together imply the conservation of critical residues in the active site (Liu et al, 2019), although extensive variations among these enzymes in the shapes of their substrate-binding sites could lead to disparate substrate preferences and deacetylation F I G U R E 4 Effects of PM_CBP active immunisation on tick acquisition and transmission. (a) Effects of PM_CBP active immunisation on tick acquisition.…”

An overall look at insect chitin deacetylases: Promising molecular targets for developing green pesticides

“…Unlike midgut CDAs, the deacetylation activity of BmCDA1 was detectable only when the cuticular chitin-binding protein CPAP-3A1 was added. 33) Taken together, the mechanism of insect CDAs to exhibit deacetylation activity was complexed and varied.…”

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

“…( Figure 2 ) [ 26 ]. Crystal structures of several fungal CDAs suggest that their substrate binding site comprises four subsites, ranging from {−2} to {+1} [ 20 , 28 , 29 ], while some bacterial CDAs appear to have less subsites [ 29 ], and one insect CDA has been shown to possess a larger number of subsites [ 30 ].…”