| Genetic and biochemical analysis of the cysteinyl residues of isopenicillin N synthase from Streptomyces clavuligerus. | |
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MedLine Citation:
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PMID: 8776857 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Isopenicillin N synthase (IPNS) from Streptomyces clavuligerus catalyses the oxidative cyclization of the acyclic tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N. All four of the cysteine residues found in this enzyme were mutated individually into serine residues, either by the polymerase chain reaction or by single-strand site-directed mutagenesis. Functional analysis of these single mutants showed that the C104S mutant lost more than 96% of its activity, while the remaining C37S, C142S, and C251S mutants each lost 30-50% of their activity. Treatment with the thiol-group-specific reagent N-ethylmaleimide confirmed the importance of the cysteine 104 residue. Activity analysis of an IPNS triple mutant (C37S, C142S, and C251S), prepared by recombining fragments of the IPNS-encoding pcbC gene from each of the three single mutants, showed that it had lost more than 90% of its activity. Conformational analysis by circular dichroism spectroscopy indicated that the IPNS triple mutant was structurally different from the wild type, suggesting that the loss of activity may be due to conformational changes rather than active site modifications. |
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Authors:
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M Durairaj; B K Leskiw; S E Jensen |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Canadian journal of microbiology Volume: 42 ISSN: 0008-4166 ISO Abbreviation: Can. J. Microbiol. Publication Date: 1996 Aug |
Date Detail:
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Created Date: 1997-03-20 Completed Date: 1997-03-20 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0372707 Medline TA: Can J Microbiol Country: CANADA |
Other Details:
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Languages: eng Pagination: 870-5 Citation Subset: IM |
Affiliation:
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Department of Biological Sciences, University of Alberta, Edmonton, Canada. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Base Sequence Cysteine / genetics Molecular Sequence Data Mutagenesis, Site-Directed Mutation Oxidoreductases / genetics*, metabolism Streptomyces / enzymology, genetics* Structure-Activity Relationship |
| Chemical | |
Reg. No./Substance:
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52-90-4/Cysteine; EC 1.-/Oxidoreductases; EC 1.21.3.1/isopenicillin N synthetase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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