Biosynthesis of polyketides the synthesis of 6-methylsalicylic acid and triacetic acid lactone in Penicillium patulum

Abstract

An enzyme fraction from P. patulum has been prepared which will synthesize 6-MSA 5 5 The abbreviations used are: NAC, N-acetylcysteamine; 6-MSA, 6-methylsalicylic acid; TAL, triacetic acid lactone. and fatty acids from acetyl CoA, malonyl CoA and NADPH. The 6-MSA synthetase was purified threefold by ammonium sulphate fractionation and catalyses the formation of 0.86 nmoles of 6-MSA per minute per mg of protein. Chemical degradation of 14C-6-MSA biosynthesized from 1,3- 14C-malonyl CoA, acetyl CoA and NADPH gave a ratio of 2.39 1 for the radioactivity in the aromatic ring to that in the terminal carboxyl group. This is consistent with the acetatepolymalonate origin of 6-MSA; the deviation from the theoretical value of 2 1 is probably due to malonyl CoA decarboxylase activity leading to the formation of 1- 14C-acetyl CoA. The rate of synthesis of 6-MSA was generally about ten times faster than the rate of fatty acid synthesis, but this ratio varied with the preparation. The 6-MSA synthetase is inhibited by isodoacetamide and N-ethymaleimide, which suggests that the synthetase activity contains sulphydryl groups in the active sites. The 6-MSA synthetase activity is inhibited by the following acetylenic thioesters: 3-pentynoyl-NAC, 3-hexynoyl-NAC and 2-hexynoyl-NAC. The activity is not inhibited by 3-hexynoic acid. The synthesis of 6-MSA can be abolished at a concentration of 10 −4 M 3-hexynoyl-NAC without affecting the synthesis of free palmitic and stearic acid. The synthesis of 6-MSA by P. patulum under in vivo conditions is also inhibited by 3-hexynoyl-NAC. The syntheses of 6-MSA and TAL (in the presence of NADPH) are inhibited to comparable extents by 5 × 10 −5 M 3-hexynoyl-NAC while the synthesis of fatty acids is not inhibited below 10 −4 M. The mechanism of 6-MSA synthesis is discussed in relation to the formation of TAL and to the possible mode of action of the acetylenic thioester inhibitors.