Comparative kinetic analysis of glycerol 3-phosphate cytidylyltransferase from Enterococcus faecalis and Listeria monocytogenes
Ashley N. Mericl, Jon A. Friesen
Med Sci Monit 2012; 18(11): BR427-434
Available online: 2012-10-31
Background: Glycerol 3-phosphate cytidylyltransferase (GCT) is an enzyme central to the synthesis of teichoic acids, components of the cell wall in gram positive bacteria. Catalysis by GCT from Enterococcus faecalis and Listeria monocytogenes has been investigated and catalytic properties compared.
Material/Methods: The genes encoding GCT were cloned from genomic DNA and recombinant proteins expressed in E. coli and purified. Enzyme assays were used to determine kinetic constants kcat and Km. Chemical crosslinking provided a means to assess quaternary structure of each GCT.
Results: Recombinant Enterococcus faecalis GCT had an apparent kcat value of 1.51 s–1 and apparent Km values of 2.42 mM and 4.03 mM with respect to substrates cytidine 5’-triphosphate (CTP) and glycerol phosphate. Listeria monocytogenes GCT had an apparent kcat value of 4.15 s–1 and apparent Km values of 1.52 mM and 6.56 mM with respect to CTP and glycerol phosphate. This resulted in kcat/Km values of 0.62 s–1mM–1 and 0.37 s–1mM–1 for E. faecalis GCT and 2.73 s–1mM–1 and 0.63 s–1mM–1 for L. monocytogenes GCT with respect to CTP and glycerol phosphate, respectively.
Conclusions: The genome of both Enterococcus faecalis and Listeria monocytogenes contain a gene that encodes a functional GCT. The genes are 67% identical at the nucleotide level and the encoded proteins exhibit a 63% amino acid identity. The purified, recombinant enzymes each appear to be dimeric and display similar kinetic characteristics. Studying the catalytic characteristics of GCT isoforms from pathogenic bacteria provides information important for the future development of potential antibacterial agents.
Keywords: Nucleotidyltransferases - metabolism, Nucleoside Diphosphate Sugars - metabolism, Molecular Sequence Data, Models, Molecular, Listeria monocytogenes - enzymology, Kinetics, Isoenzymes - metabolism, Enterococcus faecalis - enzymology, Cross-Linking Reagents - metabolism, Chromatography, Affinity, biocatalysis, Amino Acids - metabolism, Amino Acid Sequence, Protein Multimerization, Protein Structure, Quaternary, Sequence Alignment, Substrate Specificity