Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2–4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a ‘gate lock’ that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

Serratia marcescens , rod-shaped Gram-negative bacteria is classified as an opportunistic pathogen in the family Enterobacteriaceae. It causes a wide variety of infections in humans, including urinary, respiratory, ocular lens and ear infections, osteomyelitis, endocarditis, meningitis and septicemia. Unfortunately, over the past decade, antibiotic resistance has become a serious health care issue; the effective means to control and dissemination of S. marcescens resistance is the need of hour. The whole genome sequencing of S. marcescens FGI94 strain contains 4434 functional proteins, among which 690 (15.56%) proteins were classified under hypothetical. In the present study, we applied the power of various bioinformatics tools on the basis of protein family comparison, motifs, functional properties of amino acids and genome context to assign the possible functions for the HPs. The pseudo sequences (protein sequence that contain ≤100 amino acid residues) are eliminated from the study. Although we have successfully predicted the function for 483 proteins, we were able to infer the high level of confidence only for 108 proteins. The predicted HPs were classified into various classes such as enzymes, transporters, binding proteins, cell division, cell regulatory and other proteins. The outcome of the study could be helpful to understand the molecular mechanism in bacterial pathogenesis and also provide an insight into the identification of potential targets for drug and vaccine development.

Lymphatic filariasis is a debilitating vector borne parasitic disease that infects human lymphatic system by nematode Brugia malayi. Currently available anti-filarial drugs are effective only on the larval stages of parasite. So far, no effective drugs are available for humans to treat filarial infections. In this regard, aspartate semialdehyde dehydrogenase (ASDase) in lysine biosynthetic pathway from Wolbachia endosymbiont Brugia malayi represents an attractive therapeutic target for the development of novel anti-filarial agents. In this present study, molecular modeling combined with molecular dynamics simulations and structure-based virtual screening were performed to identify potent lead molecules against ASDase. Based on Glide score, toxicity profile, binding affinity and mode of interactions with the ASDase, five potent lead molecules were selected. The molecular docking and dynamics results revealed that the amino acid residues Arg103, Asn133, Cys134, Gln161, Ser164, Lys218, Arg239, His246, and Asn321 plays a crucial role in effective binding of Top leads into the active site of ASDase. The stability of the ASDase-lead complexes was confirmed by running the 30 ns molecular dynamics simulations. The pharmacokinetic properties of the identified lead molecules are in the acceptable range. Furthermore, density functional theory and binding free energy calculations were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-filarial agents to combat the pathogenecity of Brugia malayi.

Ryanodine receptors (RyRs) are homotetrameric intracellular calcium (Ca 2+ ) release channels responsible for excitation−contraction coupling of muscle cells. Diamide insecticides specifically act on RyRs of Lepidoptera and Coleoptera pests and are safe for nontargeted organisms, generating big worldwide sales. Despite their popularity, several devastating agricultural pests have been reported to be resistant to them because of mutations in a small transmembrane region of their RyRs, hinting a binding pocket nearby. A potential solution to overcome resistance is to develop new insecticides targeting different binding sites in pest RyRs. Based on a high-resolution crystal structure of diamondback moth (DBM) RyR N-terminal domain (NTD) determined by our group, we carried out extensive structure-based insecticide screening targeting the intersubunit interface. We identified eight lead compounds that selectively target the open conformation of DBM RyR, which are predicted to act as channel activators similar to diamide insecticides. Binding mode analysis shows selective binding to a hydrophobic pocket of DBM NTD-A but not to the pocket of its mammalian counterpart. We tested three available compounds on the HEK293 cell lines stably expressing DBM or mammalian RyR, one of which shows good potency and selectivity against DBM RyR. The insecticidal effect of the compound was also confirmed using fruit flies. The detailed binding mode, toxicity, absorption, distribution, metabolism, and excretion, and reactivity of the compound were predicted by bioinformatic methods. Together, our study lays a foundation for developing a new class of selective RyR-targeting insecticides to control both wild-type and resistant pests.

: Ryanodine receptor (RyR) is one of the primary targets of commercial insecticides. The diamide insecticide family, including flubendiamide, chlorantraniliprole, cyantraniliprole, etc, targets insect RyRs and can be used to control a wide range of destructive agricultural pests. The diamide insecticides are highly selective against lepidopteran and coleopteran pests with relatively low toxicity for non-target species, such as mammals, fishes, and beneficial insects. However, recently mutations identified on insect RyRs have emerged and caused resistance in several major agricultural pests throughout different continents. This review paper summarizes the recent findings on structure and function of insect RyRs as insecticide target. Specifically, we examine the structures of RyRs from target and non-target species, which reveals the molecular basis for insecticide action and selectivity. We also examine the structural and functional changes of RyR caused by the resistance mutations. Finally, we examine the progress in RyR structure-based insecticide design, and discuss how this might help the development of new generation of green insecticides.