Characterization of putative PLP-dependent beta C-S lyase from Corynebacterium diphtheriae, a possible target for a new antimicrobial agent.

The continuous emergence of antibiotic resistance in microbial pathogens requires a sustained effort to identify new antimicrobial compounds and targets. The biosynthesis of methionine is an attractive target given its importance in protein and DNA metabolism. Moreover, most of the steps in this pathway are absent in mammals, lessening the opportunity of unwanted side effects. Herein, detailed biochemical characterization of a putative pyridoxal 5’-phosphate (PLP)-dependent beta C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in Escherichia coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters and ligand-induced spectral transitions of the recombinant protein by a combination of UV/Vis and fluorescence spectroscopy. The 3D structure of beta C-S lyase from Corynebacterium diphtheriae has already been solved at 1.99 Å resolution (Joint Center for Structural Genomics). The enzyme is a homodimer composed of ~42 kDa subunits, each associated with one molecule of PLP. Structural comparison of beta C-S lyase from Corynebacterium diphtheriae with beta C-S lyase from Streptococcus anginosus1 and cystalysin from Treponema denticola2 indicates a similarity in overall folding and active site residues. We used site-directed mutagenesis to highlight the importance of the active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties and spectra of enzyme-ligand complexes. Better understanding of the active site of Corynebacterium diphtheriae beta C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors, as antibacterial therapeutics.