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Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive
D R Sylvester1, E Alvarez, A Patel
1Department of Anti-Infective Research, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, Collegeville, PA 19426, USA.
Abstract:
The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5 kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463 nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.
Insights
We characterized the UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Streptococcus pneumoniae. This enzyme is crucial for bacterial cell wall synthesis and a potential target for new antibacterial drugs.
Area of Science:
- Biochemistry
- Microbiology
- Structural Biology
Background:
- The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is essential for bacterial cell wall biosynthesis.
- Streptococcus pneumoniae is a significant Gram-positive pathogen, making its essential enzymes attractive targets for antibacterial drug discovery.
Purpose of the Study:
- To identify and characterize the MurB enzyme from Streptococcus pneumoniae.
- To provide a foundation for developing novel antibacterial agents targeting MurB.
Main Methods:
- Cloning the MurB gene into a pET28a+ expression vector and overexpressing it in E. coli.
- Purifying the recombinant protein using a two-step process.
- Spectroscopic and kinetic characterization of the purified enzyme.
Main Results:
- The S. pneumoniae MurB enzyme shares 31% identity with E. coli MurB, retaining key catalytic and binding residues.
- Overexpressed MurB was largely insoluble, but methods were optimized to yield 10% soluble protein.
- Purified MurB is a flavoprotein active in reducing UDP-N-acetylglucosamine enolpyruvate and oxidizing NADPH, exhibiting properties similar to E. coli MurB.
Conclusions:
- The characterization of S. pneumoniae MurB provides critical insights into its function and structure.
- This study establishes MurB as a promising target for the development of new antibacterial therapies against Gram-positive pathogens.