Structures and mechanism of chitin synthase and its inhibition by antifungal drug Nikkomycin Z.
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ABSTRACT:
SUBMITTER: Wu Y
PROVIDER: S-EPMC9726829 | biostudies-literature | 2022 Dec
REPOSITORIES: biostudies-literature
Publications
Structures and mechanism of chitin synthase and its inhibition by antifungal drug Nikkomycin Z.
Wu Yanan Y Zhang Min M Yang Yizheng Y Ding Xuyang X Yang Ping P Huang Kai K Hu Xinlin X Zhang Mingjie M Liu Xiaotian X Yu Hongjun H
Cell discovery 20221206 1
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Project description:The amino sugar N-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen Candida albicans. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc. Microarray analysis of gene expression was carried out under four different conditions in order to characterize the transcriptional changes induced by GlcNAc. The most highly induced genes include those that encode a GlcNAc transporter (NGT1) and the GlcNAc catabolic enzymes (HXK1, DAC1, and NAG1). GlcNAc also activated most of the genes whose expression is increased when cells are triggered with other stimuli to form hyphae. Surprisingly, GlcNAc also induced a subset of genes that are regulated by galactose (GAL1, GAL7, and GAL10), which may be due to cross talk between signaling pathways. A novel GlcNAc-induced gene, GIG1, which is not essential for GlcNAc catabolism or the induction of hyphae, was identified. However, a Gig1-green fluorescent protein (GFP) fusion protein was specifically induced by GlcNAc, and not by other sugars. Gig1-GFP localized to the cytoplasm, where GlcNAc metabolism occurs. Significantly, a gig1Δ mutant displayed increased resistance to nikkomycin Z, which inhibits chitin synthase from converting UDP-GlcNAc into cell wall chitin. Gig1 is highly conserved in fungi, especially those that contain GlcNAc catabolic genes. These results implicate Gig1 in GlcNAc metabolism.
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Project description:Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain.Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 microM - 10 microM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges.Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.
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Project description:Chitin is an essential component of the fungal cell wall. Chitin synthases (Chss) catalyze chitin formation and translocation across the membrane and are targets of antifungal agents, including nikkomycin Z and polyoxin D. Lack of structural insights into the action of these inhibitors on Chs has hampered their further development to the clinic. We present the cryo-EM structures of Chs2 from Candida albicans (CaChs2) in the apo, substrate-bound, nikkomycin Z-bound, and polyoxin D-bound states. CaChs2 adopts a unique domain-swapped dimer configuration where a conserved motif in the domain-swapped region controls enzyme activity. CaChs2 has a dual regulation mechanism where the chitin translocation tunnel is closed by the extracellular gate and plugged by a lipid molecule in the apo state to prevent non-specific leak. Analyses of substrate and inhibitor binding provide insights into the chemical logic of Chs inhibition, which can guide Chs-targeted antifungal development.
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Project description:Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc) and Mg(++) . The optimal pH was about 6.5-7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.
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Project description:Pathogenic fungi convert chitin to chitosan to evade plant perception and disarm chitin-triggered immune responses. Whether plants have evolved factors to counteract this evasion mechanism remains obscure. Here, we decipher the mechanism underlying the antifungal activity of maize secretory mannose-binding cysteine-rich receptor-like secreted protein (CRRSP), antifungal protein 1 (AFP1). AFP1 binds to multiple sites on the surface of sporidial cells, filaments, and germinated spores of the biotrophic fungus Ustilago maydis. It inhibits cell growth and budding, as well as spore germination. AFP1 promiscuously interacts with most chitin deacetylases (CDAs) by recognizing the conserved NodB domain to interfere with the enzyme activity. Deletion of O-mannosyltransferase 4 decreases protein mannosylation, which correlates with reduced AFP1 binding and antifungal activity, suggesting that AFP1 interacts with mannosylated proteins to exhibit an inhibitory effect. AFP1 also has extended inhibitory activity against Saccharomyces cerevisiae; however, AFP1 did not reduce binding to the double ΔΔcda1,2 mutant, suggesting the targets of AFP1 have expanded to other cell surface glycoproteins, probably facilitated by its mannose-binding property. Increasing chitin levels by modulating the activity of cell surface glycoproteins is a universal feature of AFP1 interacting with a broad spectrum of fungi to inhibit their growth. IMPORTANCE Plants alert immune systems by recognizing the fungal pathogen cell wall component chitin via pattern recognition cell surface receptors. Successful fungal pathogens escape the perception by deacetylating chitin to chitosan, which is also necessary for fungal cell development and virulence. Targeting glycoproteins that are associated with regulating chitin metabolism and maintaining cell wall morphogenesis presents an effective strategy to combat fungal pathogens by simultaneously altering cell wall plasticity, activating chitin-triggered immunity, and impairing fungal viability. Our study provides molecular insights into a plant DUF26 domain-containing secretory protein in warding off a broad range of fungal pathogens by acting on more than one glycoprotein target.
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