Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme
| Autor: | E. N. G. Marsh, Steven G. Kendrew, D. A. Hopwood |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1997 |
| Předmět: |
Naphthacenes
Recombinant Fusion Proteins Gene Expression Anthraquinones medicine.disease_cause Microbiology Streptomyces Actinorhodin Catalysis Substrate Specificity chemistry.chemical_compound Open Reading Frames Biosynthesis medicine Escherichia coli Histidine Molecular Biology chemistry.chemical_classification biology Streptomyces coelicolor Monooxygenase biology.organism_classification Anti-Bacterial Agents Kinetics Enzyme chemistry Biochemistry Multigene Family Mutation Oxygenases Research Article |
| Zdroj: | ResearcherID |
| Popis: | The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis. |
| Databáze: | OpenAIRE |
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