Structural basis for directional chitin biosynthesis

Abstract

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.

Comparative Transcriptome Analysis Unravels the Response Mechanisms of Fusarium oxysporum f.sp. cubense to a Biocontrol Agent, Pseudomonas aeruginosa Gxun-2

“…The four genes encoding chitin synthase in the amino sugar and nucleoside sugar metabolic pathways were downregulated, and chitin synthase is a glycosyltransferase that catalyzes the synthesis of chitin [ 36 ]. The chitin-biosynthesis capability of FOC TR4 was decreased according to the downregulation of several genes.…”

Customized chitooligosaccharide production—controlling their length via engineering of rhizobial chitin synthases and the choice of expression system

“…While the cytosolic part of the enzyme contains conserved motifs and is thus similar to other crystallized glycosyltransferases, no closely related crystallized template was available for the transmembrane part ( Figure 2 ). Even though crystal structures for two chitin synthases were recently published ( Chen et al, 2022 ; Ren et al, 2022 ), this new data could not improve our model: both chitin synthases show an even lower sequence identity to NodC_Sm and NodC_GRH2 than the cellulose synthase BcsA (see Supplementary Table S1 ), and while the nine motifs discussed by Chen et al are strongly conserved between the chitin polymer synthases of oomycetes, fungi, and arthropods, this applies to a lesser extent only for the rhizobial NodC chitin oligomer synthases. Especially the transmembrane parts of NodCs and the chitin polymer synthases differ from each other, as shown representatively for the chitin synthase from Candida albicans and NodC_GRH2 and NodC_Sm in Supplementary Figure S4 .…”

“…Dorfmueller et al have presented an analysis of NodC_Sm’s general structure and mechanism of chitin synthesis, in the process creating and thoroughly validating a structural model based on a bacterial cellulose synthase ( Dorfmueller et al, 2014 ). In addition, the first crystal structures of two chitin synthases were recently published ( Chen et al, 2022 ; Ren et al, 2022 ), providing potential alternative templates for NodC modelling.…”

Chitin Biosynthesis in Aspergillus Species

“…Recently, structural and functional data based on Chs1 from Phytophthora soybean , a pathogenic oomycete that causes root and stem rot in soybean, were found to contribute to the mechanistic understanding of chitin biosynthesis at the atomic level [ 64 ]. The analysis of Ps Chs1 by cryo-electron microscopy (cryo-EM) identified five structures that reflected different states of the enzyme: apo, linked to the substrate (UDP-GlcNAc), catalytic state, linked to the nascent chitin oligomer, and the product release state.…”

“…Finally, after many reactions, the enzyme changes its conformation to the “post-released state”, where the chitin chain is released from the CS. Subsequently, the loop gate blocks the chitin-translocated channel again, until a new substrate comes, re-starting the cycle of chitin chain biosynthesis [ 64 ].…”