MOLECULAR CHARACTERIZATION AND CLONING OF AN ESTERASE WHICH INACTIVATES THE MACROLIDE TOXIN BREFELDIN-A

The macrolide antibiotic brefeldin A (BFA) was described as a phytotox in and pathogenicity factor from Alternaria carthami Chowdhury, the ca usal agent of a devastating blight disease in safflower (Carthamus tin ctorius L.). The toxin is known to inhibit the endoplasmic reticulum-G olgi flux and processing. Conventional breeding of safflower for resis tance to the Alternaria blight disease has failed, and in situ detoxif ication of brefeldin A is a novel approach for the protection of field -grown safflower plants from the blight disease. As a first step towar ds this goal, a strain of Bacillus subtilis had been isolated which is capable of hydrolyzing brefeldin A to a non-toxic metabolite, brefeld in A acid. The BFA esterase was purified to homogeneity from B. subtil is extracts and shown to consist of a monomeric peptide of approximate ly 40 kDa. Besides brefeldin A, the esterase hydrolyzed ethyl valerate , which structurally resembles the lactone portion in the brefeldin A molecule, but failed to accept other macrolides such as erythromycin o r zearalenone. Roughly 12% of the esterase sequence was identified by microsequencing tryptic peptides and the N terminus, which lacked a me thionine leader residue. Corresponding oligonucleotide probes were emp loyed to clone the esterase gene in pUC18. One of seven clones was seq uenced and shown to code for the full size esterase protein of 372 ami no acid residues. The esterase gene was subcloned in pT7-7 and express ed in Escherichia coli yielding a fusion protein with a specific ester ase activity 3-fold over that of the enzyme purified from B. subtilis. The cloned esterase provides the basis for the generation of transgen ic safflower plants as a valuable asset in the research on nonspecific phytotoxins and in supporting breeding for Alternaria blight resistan ce.