Biosynthese von 6‐Methylsalicylsäure

Abstract

An extract was prepared from Penicillium patulum grown in submerged culture, which converted acetyl‐CoA and malonyl‐CoA into 6‐methylsalicylic acid in the presence of NADPH. The enzyme was very labile in the crude extract, but appeared stable after acetone precipitation. A 100‐fold purification was achieved by ammonium sulfate fractionation, sedimentation in the ultracentrifuge, and sucrose‐density gradient centrifugation. The enzyme was a single particle with a molecular weight of 1.1–1.5 × 10 6 as determined by gradient centrifugation with catalase and fatty acid synthetase as standards. In the first steps of purification, fatty acid synthetase from Penicillium patulum migrated with the methylsalicylic acid synthetase complex. A separation of the two enzyme systems was accomplished by sucrose‐density gradient centrifugation. The purified enzyme represents a new multienzyme complex which shares several common properties with fatty acid synthetase. Methylsalicylic acid synthetase is inhibited by iodoacetamide and N ‐ethylmaleinimide. The incubation of enzyme with acetyl‐CoA and malonyl‐CoA in the absence of NADPH led to triacetic acid lacton. This indicates that in 6‐methylsalicylic acid synthesis reduction occurs at the triacetic acid level before the final condensation with malonyl‐CoA. Triacetic acid lacton was produced by the enzyme at 10% of the rate of 6‐methylsalicylic acid synthesis in the complete system. This rate of lacton synthesis, however, is an order of magnitude greater than that catalysed by fatty acid synthetase. A possible explanation for this difference in behavior is given. Covalent linkage of substrates and intermediates to SH‐groups of the protein during the synthesis of 6‐methylsalicyslic acid is suggested. A reaction scheme leading to 6‐methylsalicylic acid is proposed and the partial reactions of this synthesis are discussed with respect to similar reactions of fatty acid biosynthesis.