KeywordStructural basis for directional chitin biosynthesis

The biosynthesis of chitin is essential for the survival and reproduction of various organisms from different taxonomic groups, such as life-threatening fungi, agriculture-devastating oomycetes and insect pests. Therefore, it provides a preferred target for discovering antifungal agents or pesticides.

The core of the chitin biosynthetic machinery is an integral membrane enzyme named chitin synthase (CHS) (EC 2.4.1.16). CHS belongs to glycosyltransferase family 2 (GT2), a large enzyme family that includes cellulose, alginate and hyaluronan synthases. Chitin synthesis is proposed to involve three major steps: (1) the processive addition of GlcNAc from UDP-GlcNAc (donor substrate) to the terminal C4-hydroxyl group of the nascent chitin chain (acceptor substrate) by the catalytic domain of the enzyme facing the cytoplasmic side; (2) the release of the nascent chain to the extracellular space through a transmembrane channel within the enzyme; and (3) the spontaneous assembly of released nascent chains into nanofibrils. CHS controls the first two steps of this process but may also participate in the formation of fibrils. Despite differing in the number of transmembrane helices and organization of the respective cytosolic domains, CHSs from various species share a conserved catalytic domain (Extended Data Fig. 1), thus allowing the development of competitive inhibitors with broad-spectrum activities. Because chitin is absent in plants and mammals, CHS might constitute one of the safest among the 30 currently used insecticidal and fungicidal targets for the control of fungal pathogens and insect pests. Among the fungicidal agents that target CHS is nikkomycin Z (NikZ), which consists of a pyrimidine-nucleoside peptide backbone and is a first-generation broad-spectrum CHS inhibitor currently in phase II clinical trials.

P. sojae is a pathogen that causes soybean (Glycine max L.) root and stem rot, which results in economic losses of more than US$1 billion per year. Knockout of the P. sojae chitin synthase PsChs1 impairs mycelial growth, sporangial production and zoospore release, and thus greatly reduces the virulence of P. sojae. PsChs1 serves as both an excellent antifungal target and a model system for CHS research. In this study, we report five cryo-electron microscopy (cryo-EM) structural snapshots of PsChs1, which provide not only a mechanistic understanding of chitin biosynthesis at the atomic level but also a structural basis for the rational design of CHS-targeting inhibitors.

PsChs1 was mainly purified as a dimer in solution (Extended Data Fig. 2a–c). Activity of PsChs1 clearly depends on specific divalent ions, and EDTA completely blocked enzyme activity (Extended Data Fig. 2d). The addition of GlcNAc together with divalent ions significantly increased the activity of PsChs1 (Extended Data Fig. 2d). Enzyme kinetics revealed a Hill coefficient of 1, indicating that GlcNAc is not a positive effector of PsChs1 (Extended Data Fig. 2e). In line with previous data, which has shown that yeast chitin synthase Chs2 can use 2-acylamido analogues of GlcNAc as acceptors of GlcNAc derived from UDP-GlcNAc, this finding suggests that free GlcNAc may act as an acceptor to prime the reaction. Of note, the addition of (GlcNAc)2–5 did not affect enzyme activity (Extended Data Fig. 2f).

The sugar polymer produced by PsChs1 could be degraded by Ostrinia furnacalis Chi-h, a chitinase that specifically hydrolyses chitin, confirming that the product is chitin (Extended Data Fig. 2d). Using a scanning electron microscope, we observed that the synthesized chitin appeared as a fibrous material, and the amount of chitin fibre increased as the reaction time progressed (Extended Data Fig. 2g). Under a confocal laser scanning microscope, chitin was specifically detected by wheat germ agglutinin coupled to the fluorophore fluorescein isothiocyanate. It appeared as aggregated fibrillar material at high magnification by a scanning electron microscope, but as a ‘roundish’ soft material at lower magnification by a confocal laser scanning microscope (Extended Data Fig. 2h). The isomorphic type of the synthesized chitin was determined by X-ray diffraction and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy (Extended Data Fig. 2i). As indicated in ATR-FTIR spectroscopy, the synthesized chitin showed the same adsorption spectrum as shrimp α-chitin, where the characteristic C=O stretching (amide I) band at 1,620–1,670 cm−1 appeared as a doublet, which was clearly distinguishable from a singlet that appeared in the crystalline β-chitin. The ATR-FTIR spectra are in line with the X-ray diffraction results. The four sharp diffraction peaks of synthesized chitin observed at 9.3°, 12.7°, 19.3° and 26.4°, which corresponded to the 020, 021, 110 and 013 planes, respectively, are typical crystal patterns of α-chitin. Therefore, the polymer synthesized by PsChs1 is α-chitin, which is consistent with data in the literature that have demonstrated that chitin formed in oomycete species is of the α-type.

The different cryo-EM structures of PsChs1 were reconstructed by imposing a C2 symmetry and reached overall resolutions of 3.1 Å (UDP bound), 3.2 Å (NikZ bound), 3.3 Å (apo and UDP-GlcNAc bound) and 3.9 Å (UDP/(GlcNAc)3bound) (Extended Data Table 1 and Extended Data Figs. 3–7). The EM maps were of sufficient quality to allow de novo building of residues 40–860, with a disordered region of residues 743–758, in all five structures. The donor substrate-bound structure shows an additional N-terminal region from residues 23–39 (Extended Data Fig. 4e). All the structures include the N-terminal domain (NTD), the glycosyltransferase (GT) domain and all α-helices of the C-terminal transmembrane (TM) domain (Fig. 1a–c). The TM region comprises a cluster of six TM helices (TM1–6) that reside on top of three amphipathic interface helices (IF1–3) located at the boundary between the membrane and the cytosol (Fig. 1c and Extended Data Fig. 8a). Although TM topology algorithms predict IF3 to form a TM helix (Extended Data Fig. 8b), our structures revealed that it actually forms a bent helix parallel to the membrane, as suggested previously for Chs3 in yeast24. TM5 is an extraordinarily long helix (approximately 80 Å in length) that spans from the TM domain to the cytosolic region and projects into the opposite protomer like a sword (Fig. 1b).

a,b, The structure of the PsChs1 dimer is shown in surface (a) and ribbon (b) representations as viewed from the extracellular side of the membrane (top view), within the plane of the membrane (side view), or the cytoplasmic side (bottom view). The approximate position of the membrane is marked with grey shading, and the presumed chitin-translocating channel is marked with arrows. The TM helices, GT domain, IF helices, LG subdomain, MIT subdomain and SP subdomain of one protomer are coloured blue, violet, pink, green, purple and light grey, respectively. The other protomer is coloured yellow. The unresolved region (residues 743–758) is shown as dashed lines. c, Domain architecture and ribbon representation of a PsChs1 protomer in two orientations. d, Sliced-surface view of the presumed chitin-translocating channel. Pro454 and Trp539 are at the channel entrance and are highlighted in red and pink, respectively. e, The reaction chamber of PsChs1 (left) and the conserved motifs that constitute the reaction chamber (right) are shown. The uridine-binding tub, catalytic cave and entrance of the chitin-translocating channel are coloured grey, blue and red, respectively. Residues that are important for enzyme activity are underlined and represented as sticks.