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Characterization of chitin synthase from Botrytis cinerea
B E Causier1, R J Milling, S G Foster
1Department of Microbiology, University of Leeds, UK.
Microbiology (Reading, England)
|September 1, 1994
Summary
This study investigates chitin synthase in Botrytis cinerea, finding that cytosol significantly activates the enzyme and identifying two gene fragments, CHS1 and CHS2. CHS1 shows similarity to Neurospora crassa CHS1.
Area of Science:
- Biochemistry
- Molecular Biology
- Mycology
Background:
- Chitin synthase is a key enzyme in fungal cell wall biosynthesis.
- Understanding its regulation and genetic basis is crucial for developing targeted antifungal strategies.
Purpose of the Study:
- To characterize the kinetic properties and regulation of chitin synthase in Botrytis cinerea.
- To identify and analyze chitin synthase genes in Botrytis cinerea.
Main Methods:
- Enzyme kinetics assays using microsomal preparations.
- Inhibition studies with nikkomycin Z, polyoxin D, and edifenphos.
- Enzyme activation studies with trypsin and cytosol.
- Degenerate PCR amplification and database homology searches for chitin synthase genes.
Main Results:
- Chitin synthase exhibited competitive inhibition by nikkomycin Z and polyoxin D, and non-competitive inhibition by edifenphos.
- Microsomal chitin synthase activity was significantly stimulated by preincubation with cytosol (20-fold increase) and trypsin (4-fold increase).
- Protease inhibitors, particularly phenylmethanesulphonyl fluoride and chymostatin, blocked cytosol-mediated activation.
- Two chitin synthase gene fragments (CHS1 and CHS2) were amplified, with CHS1 showing high similarity to the Neurospora crassa CHS1 gene.
Conclusions:
- Cytosol-derived proteases play a significant role in activating Botrytis cinerea chitin synthase.
- The identification of CHS1 provides a molecular basis for further genetic studies of chitin synthesis in this fungus.