Chorismate synthase from Staphylococcus aureus

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The Staphylococcus aureus aroC gene encodes chorismate synthase, essential for folate biosynthesis.
• The Staphylococcus aureus ndk gene encodes nucleoside diphosphate kinase (Ndk), involved in nucleotide metabolism.
• Gene organization and function in S. aureus are crucial for understanding bacterial physiology.

Purpose of the Study:

• To clone and characterize the aroC and ndk genes from Staphylococcus aureus.
• To investigate the genetic organization and linkage of aroC, ndk, and gerC genes.
• To overexpress, purify, and characterize Staphylococcus aureus chorismate synthase.

Main Methods:

• Gene cloning and DNA sequencing of aroC and ndk.
• Southern hybridization for gene mapping.
• Overexpression in Escherichia coli using a T7 promoter.
• Enzyme purification and characterization (mass spectrometry).

Main Results:

• The aroC gene, encoding a 388-amino acid protein with 61% identity to Bacillus subtilis AroF, was identified.
• The ndk gene, encoding a 149-amino acid protein highly identical to other bacterial Ndk, was characterized.
• Linkage of gerC genes with aroC and ndk was observed, similar to B. subtilis.
• Purified S. aureus chorismate synthase is a homotetramer with a subunit molecular mass of 43024 Da.

Conclusions:

• The genetic organization of aroC, ndk, and gerC genes in S. aureus is conserved with B. subtilis.
• Staphylococcus aureus chorismate synthase has been successfully overexpressed, purified, and characterized.
• Comparative analysis of S. aureus chorismate synthase properties provides insights into enzyme function across species.