THE SUBCELLULAR ORGANIZATION OF ITACONATE BIOSYNTHESIS IN ASPERGILLUS-TERREUS

A novel assay method for cis-aconitate decarboxylase (EC 4.1.1.6) has been developed. Differential centrifugation of cell-free extracts of Aspergillus terreus prepared by Novozyme treatment/nitrogen cavitation of itaconate-producing mycelia has shown that cis-aconitate decarboxylase, a key enzyme of itaconate biosynthesis, is localized exclusively in the cytosol. The intracellular localization and maximal activities of nine other enzymes putatively involved in itaconate biosynthesis were examined under both producing and non-producing growth conditions. Two striking differences between growth and production mycelia were observed: (a) cis-aconitate decarboxylase was absent from growing mycelia, and (b) aconitase activity was increased at least two-fold in production mycelia. No significant variations in the maximal activities or intracellular localization were observed for the other enzymes investigated. Mitochondrial and cytosolic isoenzymes of NADP: isocitrate dehydrogenase and malate dehydrogenase were detected by cellulose acetate strip electrophoretic analysis, whereas this analysis detected only single isoenzymes of aconitase and citrate synthase. We therefore propose that in itaconate accumulation cis-aconitate is synthesized in the mitochondrion and then transported to the cytosol where it is converted to itaconate by cis-aconitate decarboxylase.