f Inhibition of purified isocitrate lyase identified itaconate and oxalate as potential antimetabolites for the riboflavin overproducer Ashbya gossypii
- Authors: Georg Schmidt, K.-Peter Stahmann, Hermann Sahm
- Author for correspondence: K.-Peter Stahmann. Tel: +49 2461 612843. Fax: +49 2461 612710.
- Microbiology, February 1996 142: 411-417, doi: 10.1099/13500872-142-2-411
- Subject: Biotechnology
- Published Online:
Summary: A specific isocitrate lyase (ICL) activity of 0.17 U (mg protein)−1 was detected in cultures of the riboflavin-producing fungus Ashbya gossypii during growth on soybean oil. Enzyme activity was not detectable during growth on glucose [<0.005 U (mg protein)−1], indicating a regulation. The enzyme was purified 108-fold by means of ammonium sulphate fractionation, gel filtration and cation-exchange chromatography. SDS-PAGE of the purified protein showed a homogeneous band with an M r of 66000. The M r of 254000 determined by gel-filtration chromatography indicated a tetrameric structure of the native protein. The enzyme was found to have a pH optimum for the isocitrate cleavage of 7.0, and the K m for threo-DL-isocitrate was determined as 550 μ. Enzyme activity was Mg2+− dependent. In regulation studies ICL was weakly inhibited by central metabolites. A concentration of 10 mM phosphoenolpyruvate or 6-phosphogluconate revealed a residual activity of more than 40%. On the other hand, oxalate (K i: 4 μM) and itaconate (K i: 170 μM) showed a strong inhibition and may therefore be interesting as antimetabolites.
© Society for General Microbiology 1996 | Published by the Microbiology Society
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