Studies of the biosynthesis of tentoxin by Alternaria alternata

Biosynthesis of the phytotoxin, tentoxin, its regulation and the enzymic synthesis steps were studied in vivo and in vitro. The physiology of biosynthesis of tentoxin in vivo was investigated by using sections of mycelial mats incubated in buffer. Differentiated mycelia could be studied under defined conditions. The de novo synthesis of tentoxin was measured by incorporation of [U-14C]leucine into tentoxin. The investigation system was stable for 10 h. Biosynthesis and the growth of biomass started before day 5 of culture, with the maximum between days 9 and 12. After this, biosynthesis quickly declined, pH values about 7 were optimal, and pH values above and below this led to an increased release of tentoxin stored in the cells. The formation of tentoxin by older mycelia was not regulated by acetate, phosphate or glucose, which was not utilized. Precursor amino acids, applied at the start of the culture, slightly activated the synthesis of tentoxin. Older mycelia were inhibited. Substances from the host plant (Brassica chinensis) reduced the de novo synthesis of tentoxin. Enzyme separation studies suggested that biosynthesis of tentoxin involves a multienzyme (greater than or equal to 400 kDa), which is a polyfunctional protein without subunits. Experiments suggested that the synthetase contains active SH-groups and an integrated activity of methyltransferase. The precursor amino acids are activated by ATP and bound at the enzyme. N-Methylation occurs with the enzyme-bound amino acids or during the elongation of the growing peptide chain. Methionine is the primary donor of the methyl groups, but the immediate methylation reaction needs S-adenosyl methionine (SAM). The methylation is essential for the continuation of biosynthesis. The elongation proceeds either stepwise from glycine by binding alanine/methylalanine, phenylalanine/methylphenylalanine and leucine or by formation and linkage of two dipeptides glycine-alanine/methylalanine and phenylalanine/methylphenylalanine-leucine. At the end of this process dihydrotentoxin, the direct precursor of tentoxin, is released from the synthetase probably by cyclization. Independent of this first enzyme, dihydrotentoxin is transformed into tentoxin. This last reaction step is reversible. The rate of transformation of dihydrotentoxin to tentoxin is higher, but in this direction the native turnover is relatively low. The synthesis of tentoxin probably occurs in a manner similar to other well-known cyclic peptides via a 'thiotemplate mechanism'; the highest enzyme activity in vitro occurs between days 9 and 11 of culture at a pH value of 7.