CHARACTERIZATION OF CHITIN SYNTHASE FROM BOTRYTIS-CINEREA

Chitin synthase in a microsomal preparation from Botrytis cinerea had an apparent K-m for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent K-i values of approximately 0.1 mu M and 6 mu M respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (K-iapp 54 mu M). Preincubation of microsomes for 2 h at 25 degrees C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (15 mu g ml(-1)) or cytosol (350 mu g cytosolic protein ml(-1)) for 10 min at 25 degrees C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.