Molecular characterization and cloning of an esterase which inactivates the macrolide toxin brefeldin A.

Abstract

The macrolide antibiotic brefeldin A (BFA) was described as a phytotoxin and pathogenicity factor from Alternaria carthami Chowdhury, the causal agent of a devastating blight disease in safflower (Carthamus tinctorius L.). The toxin is known to inhibit the endoplasmic reticulum-Golgi flux and processing. Conventional breeding of safflower for resistance to the Alternaria blight disease has failed, and in situ detoxification of brefeldin A is a novel approach for the protection of field-grown safflower plants from the blight disease. As a first step towards this goal, a strain of Bacillus subtilis has been isolated which is capable of hydrolyzing brefeldin A to a non-toxic metabolite, brefeldin A acid. The BFA esterase was purified to homogeneity from B. subtilis extracts and shown to consist of a monomeric peptide of approximately 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 or zearalenone. Roughly 12% of the esterase sequence was identified by microsequencing tryptic peptides and the N terminus, which lacked a methionine leader residue. Corresponding oligonucleotide probes were employed to clone the esterase gene in pUC18. One of seven clones was sequenced and shown to code for the full size esterase protein of 372 amino acid residues. The esterase gene was subcloned in pT7-7 and expressed in Escherichia coli yielding a fusion protein with a specific esterase activity 3-fold over that of the enzyme purified from B. subtilis. The cloned esterase provides the basis for the generation of transgenic safflower plants as a valuable asset in the research on nonspecific phytotoxins and in supporting breeding for Alternaria blight resistance.