Mutational Evidence Supporting the Involvement of Tripartite Residues His183, Asp185, and His243 in Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Catalysis.
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- SIM Janet
- Department of Microbiology, Faculty of Medicine, National University of Singapore
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- SIM Tiow-Suan
- Department of Microbiology, Faculty of Medicine, National University of Singapore
Bibliographic Information
- Other Title
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- Mutational Evidence Supporting the Involvement of Tripartite Residues His183, Asp185, and His243 in<i>Streptomyces clavuligerus</i>Deacetoxycephalosporin C Synthase for Catalysis
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Abstract
Deacetoxycephalosporin C synthase (DAOCS) is a non-heme iron-binding and α-ketoglutarate dependent enzyme involved in catalyzing the biosynthesis of cephalosporins and cephamycins, antibiotics more potent than penicillins. In the crystal structure complex of Streptomyces clavuligerus DAOCS (scDAOCS), it was proposed that histidine-183, aspartate-185, and histidine-243 are putative iron-binding ligands. However, coordinates proposed for crystal structures of proteins may not definitely comply with catalysis. Hence, site-directed mutagenesis was done to replace each of these amino acid residues with leucine. The constructed expression vectors bearing the mutations were found to express the respective scDAOCS mutant enzymes at high levels in Escherichia coli BL21(DE3). Through enzymatic assays, it was shown that while the wildtype enzyme could convert penicillin to a more active cephalosporin, the substitution of the three proposed iron-binding sites of scDAOCS completely abolished the same activity in the respective mutant enzymes. Thus, these results clearly indicate that histidine-183, aspartate-185, and histidine-243 of scDAOCS are essential for the ring expansion activity.<br>
Journal
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- Bioscience, Biotechnology, and Biochemistry
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Bioscience, Biotechnology, and Biochemistry 64 (4), 828-832, 2000
Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Details 詳細情報について
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- CRID
- 1390282681450412032
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- NII Article ID
- 110002679998
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- NII Book ID
- AA10824164
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- COI
- 1:CAS:528:DC%2BD3cXjtVeltrY%3D
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- ISSN
- 13476947
- 09168451
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- NDL BIB ID
- 5379972
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- PubMed
- 10830499
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- PubMed
- CiNii Articles
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- Abstract License Flag
- Disallowed