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Purification of lincosaminide O-nucleotidyltransferase from Streptomyces coelicolor Müller
V P Marshall1, J E McGee, J I Cialdella
1Upjohn Laboratories, Upjohn Company, Kalamazoo, MI 49001.
Abstract:
An enzyme (lincosaminide O-nucleotidyltransferase) that catalyzes 3-(5'-ribonucleotidylation) of pirlimycin and several other lincosaminide antibodies has been purified approximately 35-fold from cell-free extracts of Streptomyces coelicolor Müller NRRL 3532 (UC 5240). The crude enzyme was prepared using lysozyme and was treated with MnCl2 and (NH4)2SO4. Final purification was achieved by anion exchange chromatography. The pirlimycin reaction product was verified as being pirlimycin-3-(5'-adenylate) by NMR spectroscopy and MS. As a result of purification, this lincosaminide nucleotidylating and inactivating enzyme was separated from the macrolide phosphorylating enzyme also present in the cell-free extract.
Insights
Researchers purified lincosaminide O-nucleotidyltransferase from Streptomyces coelicolor. This enzyme adds ribonucleotides to pirlimycin, inactivating the antibiotic.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Lincosaminide antibiotics, such as pirlimycin, are crucial in treating bacterial infections.
- Understanding antibiotic resistance mechanisms, including enzymatic inactivation, is vital for developing new therapeutic strategies.
Purpose of the Study:
- To purify and characterize the enzyme responsible for the nucleotidylation of lincosaminide antibiotics.
- To elucidate the specific reaction catalyzed by this enzyme and its role in antibiotic inactivation.
Main Methods:
- Enzyme purification from Streptomyces coelicolor cell-free extracts using lysozyme treatment, MnCl2, (NH4)2SO4, and anion exchange chromatography.
- Characterization of the enzyme's activity and the reaction product using Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry (MS).
Main Results:
- A 35-fold purification of lincosaminide O-nucleotidyltransferase was achieved.
- The enzyme was confirmed to catalyze the 3-(5'-ribonucleotidylation) of pirlimycin, forming pirlimycin-3-(5'-adenylate).
- The purified enzyme was separated from a co-existing macrolide phosphorylating enzyme.
Conclusions:
- Lincosaminide O-nucleotidyltransferase plays a role in the inactivation of pirlimycin and related antibiotics.
- This enzymatic activity represents a potential mechanism of antibiotic resistance.
- The purification and characterization of this enzyme provide a basis for further studies into lincosaminide antibiotic metabolism and resistance.