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Does the detoxification of penicillin side‐chain precursors depend on microsomal monooxygenase and glutathione S‐transferase in Penicillium chrysogenum ?
Author(s) -
Emri Tamás,
Oláh Brigitta,
Sámi László,
Pócsi István
Publication year - 2003
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.200390032
Subject(s) - penicillium chrysogenum , glutathione , monooxygenase , biochemistry , microsome , chemistry , detoxification (alternative medicine) , cytochrome p450 , metabolism , enzyme , medicine , alternative medicine , pathology
The glutathione (GSH) S‐conjugation of 1,2‐epoxy‐3‐(4′‐nitrophenoxy)propane was catalysed predominantly by microsomal glutathione S‐transferase (mGST) in Penicillium chrysogenum . The specific mGST activity unlike the cytosolic GST (cGST) activity increased substantially when the penicillin side‐chain precursor phenoxyacetic acid (POA) was included in the culture medium. Therefore, a microsomal monooxygenase (causing possible release of epoxide intermediates) and mGST‐dependent detoxification pathway may exist for the side‐chain precursors as an alternative to microsomal activation to acyl‐CoA and subsequent transfer to β‐lactam molecules. The P. chrysogenum pahA and Aspergillus nidulans phacA gene products, which are cytochrome P450 monooxygenases and are able to hydroxylate phenylacetic acid (PA) at position 2 on the aromatic ring, are unlikely to release toxic epoxide intermediates but epoxidation of PA and POA due to the action of other microsomal monooxygenases cannot be excluded. The GSH‐dependent detoxification of POA was provoked by a well‐controlled transient lowering of pH (down to 5.0) at the beginning of the production phase in a fed‐batch fermentation system. Both the specific GST and γGT activities were increased but the intracellular GSH concentrations remained unaltered unless the pH of the feed was transiently lowered below 5.0. At pH 4.6, the GSH pool was depleted rapidly but no antibiotic production was observed. Although sucrose was taken up effectively by the cells, cell death and autolysis were progressing. Therefore, the industrial exploitation of the GSH‐dependent detoxification of penicillin side‐chain precursors to reduce intracellular GSH‐levels in order to avoid the GSH inhibition of the β‐lactam biosynthetic enzymes seems to be rather unlikely. P. chrysogenum mGST and cGST were separated using GSH‐Sepharose 6B affinity chromatography. The purified cGST possessed a homodimer ( α 2 ) tertiary structure with M r , α = 29500.