Aconitases are monomeric proteins, which are involved in the citric acid cycle as well as in the glyoxylate cycle. Beside this enzymatic function, the major aconitase of E. coli and cytoplasmatic aconitase of mammals serve as mRNA-binding posttranscriptional regulators, depending on the status of its iron-sulfur cluster. We describe a new enzymatic function associated with the naturally occurring oxidative damaged cluster of aconitase AcoA from Aspergillus nidulans, most likely the [3Fe-4S]-form. The [4Fe-4S] cluster is associated with the aconitase activity, but oxidative stress or iron-chelating agents increases a different enzymatic activity, namely the methylaconitase activity. The methylaconitase is part of the propionate-degrading methylcitrate cycle and catalyzes the reaction of methyl-cis-aconitate to methylisocitrate. However, other aconitases, e.g. from pig heart or E. coli seem to bear both activities in the [4Fe-4S]-form. In order to get a strain producing high levels of aconitase we transformed the wild type with the acoA-gene. In crude extracts of the transformant strains, we observed a 3-fold increase of both enzymatic activities compared with the wild type. At the beginning of a typical purification, the ratio of aconitase to methylaconitase was 5:1. During the purification the aconitase looses the labile 4th iron and the ratio changed to 1:9 towards methylaconitase activity at the end of the purification. Under anaerobic reducing conditions with iron and sulphur the aconitase activity was reconstituted with simultaneously diminished methylaconitase activity. EPR- and UV-Vis-Spectra of different cluster-types are presented. Additionally, the concentration of [3Fe-4S]-clusters in a reactivated probe was estimated and correlated with the activity.
Full conference title:
23rd Fungal Genetics Conference
- Fungal Genetics Conference 23rd (2002)