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Factors affecting the isopenicillin N synthetase reaction
D Perry1, E P Abraham, J E Baldwin
1Sir William Dunn School of Pathology, University of Oxford, U.K.
The Biochemical Journal
|October 1, 1988
Summary
Isopenicillin N synthetase (IPNS) requires iron and oxygen for activity. Ascorbate and glutathione enhance IPNS catalytic efficiency and stability during isopenicillin N production.
Area of Science:
- Biochemistry
- Enzymology
- Antibiotic Synthesis
Background:
- Isopenicillin N synthetase (IPNS) is crucial for antibiotic production.
- IPNS activity is dependent on Fe2+ and O2.
- Understanding factors affecting IPNS is key to optimizing isopenicillin N (IPN) synthesis.
Purpose of the Study:
- To investigate factors influencing the activity and stability of highly purified Isopenicillin N synthetase (IPNS).
- To determine the role of metal ions, reducing agents, and substrate concentration on IPNS catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV) to IPN.
Main Methods:
- Purification of IPNS from Cephalosporium acremonium.
- Enzyme assays measuring IPNS activity under various conditions.
- Use of EDTA to quench reactions and Chelex-100 resin for metal ion removal.
- Inhibition studies using thiol-blocking reagents.
Main Results:
- IPNS undergoes inactivation during LLD-ACV conversion, with reduced stability in the absence of ascorbate.
- Ascorbate and glutathione (GSH) significantly increase the turnover number of IPNS.
- Optimal Fe2+ concentration is approximately 10 microM after metal ion removal; other transition metals act as inhibitors.
- Dithiothreitol (DTT) and GSH stimulate IPNS activity, but GSH is more stable under reaction conditions.
- IPNS is inhibited by specific thiol-blocking reagents, with some inhibition reversible by DTT.
Conclusions:
- Ascorbate and GSH are essential cofactors for maintaining IPNS activity and stability during IPN biosynthesis.
- Precise control of Fe2+ concentration is critical for optimal IPNS function.
- Understanding these factors can inform strategies for enhancing antibiotic production.
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