Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis
| Autor: | Suellen Mary Samson, Thomas Dominick Ingolia, Jerry L. Chapman, Ramamoorthy Belagaje, Stephen Wyatt Queener |
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| Rok vydání: | 1987 |
| Předmět: |
Stereochemistry
Penicillium chrysogenum Serine chemistry.chemical_compound Structure-Activity Relationship Biosynthesis Amino Acid Sequence Cysteine Site-directed mutagenesis Alleles chemistry.chemical_classification Multidisciplinary biology Base Sequence Acremonium Mutagenesis biology.organism_classification Enzyme assay Amino acid Enzymes chemistry Biochemistry Gene Expression Regulation Mutation biology.protein Oxidoreductases Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America. 84(16) |
| ISSN: | 0027-8424 |
| Popis: | The predicted amino acid sequences of isopenicillin N synthetase from both Cephalosporium acremonium and Penicillium chrysogenum have two cysteine residues in analogous positions (Cys-106 and Cys-255 in the C. acremonium numbering). To examine the role of these cysteine residues in the activity of the C. acremonium enzyme, we used site-directed in vitro mutagenesis to change these cysteine residues to serine residues. Mutation of Cys-255 reduces specific activity approximately equal to 50%, whereas mutation of Cys-106 or mutation of both Cys-106 and Cys-255 reduces specific activity about 97%. This suggests that the cysteines are important but not essential for IPNS activity. Alkylation of IPNS also almost completely inactivated the enzyme, but residual activity could have been due to incomplete alkylation. Atomic substitution via genetic manipulation in this case is a more accurate means of assessing the role of sulfhydryl moieties in enzyme activity. |
| Databáze: | OpenAIRE |
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