Does the detoxification of penicillin side-chain precursors depend on microsomal monooxygenase and glutathione S-transferase in Penicillium chrysogenum?

T. Emri, Brigitta Oláh, László Sámi, I. Pócsi

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6 Citations (Scopus)

Abstract

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 (α2) tertiary structure with M r, α = 29500.

Original languageEnglish
Pages (from-to)287-300
Number of pages14
JournalJournal of Basic Microbiology
Volume43
Issue number4
DOIs
Publication statusPublished - 2003

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Penicillium chrysogenum
Aryl Hydrocarbon Hydroxylases
Glutathione Transferase
Penicillins
Lactams
Epoxy Compounds
Autolysis
Aspergillus nidulans
Propane
Acyl Coenzyme A
Poisons
Mixed Function Oxygenases
Affinity Chromatography
Sepharose
Cytochrome P-450 Enzyme System
Fermentation
Glutathione
Culture Media
Sucrose
Cell Death

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Microbiology
  • Genetics

Cite this

@article{18b6310ee63643cfb8388ac33408d05a,
title = "Does the detoxification of penicillin side-chain precursors depend on microsomal monooxygenase and glutathione S-transferase in Penicillium chrysogenum?",
abstract = "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 (α2) tertiary structure with M r, α = 29500.",
author = "T. Emri and Brigitta Ol{\'a}h and L{\'a}szl{\'o} S{\'a}mi and I. P{\'o}csi",
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T1 - Does the detoxification of penicillin side-chain precursors depend on microsomal monooxygenase and glutathione S-transferase in Penicillium chrysogenum?

AU - Emri, T.

AU - Oláh, Brigitta

AU - Sámi, László

AU - Pócsi, I.

PY - 2003

Y1 - 2003

N2 - 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 (α2) tertiary structure with M r, α = 29500.

AB - 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 (α2) tertiary structure with M r, α = 29500.

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