A Structural and Biochemical Model of Processive Chitin Synthesis

Background: Chitin synthesis is an attractive drug target in a range of organisms but is not understood at the molecular level. Results: The chitooligosaccharide synthase NodC can be assayed with a novel HTS assay, and the mechanism/fold can be probed by site-directed mutagenesis and topology mapping. Conclusion: NodC is a model system to probe chitin synthesis. Significance: This work enables the exploitation of chitin synthesis as a drug target. Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target.

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