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Cloning and expression of Staphylococcus aureus and Treptococcus pyogenes murD genes encoding uridine diphosphate
M El-Sherbeini1, W M Geissler, J Pittman
1Department of Enzymology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA. Mohamed_El-Sherbeini@merck.com
Abstract:
Bacterial UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD), a cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent addition of D-glutamate to an alanyl residue of the UDP-N-acetylmuramyl-L-alanine precursor, generating the dipeptide. The murD gene was cloned from both Staphylococcus aureus and Streptococcus pyogenes. Sequence analysis of the S. aureus murD gene revealed an open reading frame of 449 amino acids. The deduced aa sequence of S. aureus MurD is highly homologous to MurD from Escherichia coli, Haemophilus influenzae, Bacillus subtilis and St. pyogenes. Recombinant MurD protein from both S. aureus and St. pyogenes was separately overproduced in E. coli and purified as His-tagged fusion. Both recombinant enzymes catalyzed the ATP-dependent addition of D-glutamate to the precursor sugar peptide.
Insights
Researchers cloned and analyzed the murD gene from Staphylococcus aureus and Streptococcus pyogenes. Both bacterial MurD enzymes were successfully produced and purified, confirming their role in peptidoglycan synthesis.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Peptidoglycan is a crucial component of bacterial cell walls.
- UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD) is essential for peptidoglycan biosynthesis.
- MurD catalyzes the ATP-dependent addition of D-glutamate.
Purpose of the Study:
- To clone and characterize the murD gene from Staphylococcus aureus and Streptococcus pyogenes.
- To investigate the biochemical activity of recombinant MurD proteins from these bacteria.
Main Methods:
- Cloning of the murD gene from S. aureus and S. pyogenes.
- Sequence analysis of the S. aureus murD gene.
- Overproduction and purification of His-tagged recombinant MurD proteins in E. coli.
- Enzymatic assays to confirm MurD activity.
Main Results:
- The S. aureus murD gene contains an open reading frame encoding 449 amino acids.
- The deduced amino acid sequence of S. aureus MurD shows high homology to MurD proteins from other bacterial species.
- Both recombinant S. aureus and S. pyogenes MurD enzymes were successfully purified.
- Purified recombinant MurD enzymes demonstrated ATP-dependent activity in catalyzing D-glutamate addition.
Conclusions:
- The murD gene from S. aureus and S. pyogenes encodes functional peptidoglycan ligase enzymes.
- The characterized MurD proteins are potential targets for antibacterial drug development.
- Homology among MurD proteins suggests conserved mechanisms in peptidoglycan synthesis across different bacterial species.