Exploration of the Active Site of Corynebacterium diphtheriae C-S Lyase, a Possible Target for New Antimicrobial Drug

The emergence of resistance to antibacterial agents is a pressing concern for human health. New drugs to combat this problem are therefore in great demand. Acquired bacterial resistance has caused several antibiotics to become useless or, at best, compromised in their ability to counteract bacterial infection [Coates et al., 2002]. The manifestation of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The potential of amino acid biosynthesis as an antimicrobial target has been validated both chemically and biochemically [Ejim et al., 2007]. Methionine represents a key amino acid in prokaryotes and it is an attractive antimicrobial target because of its important role in cell metabolism. Methionine, in the form of S-adenosylmethionine, is the methyl donor for a number of essential biochemical reactions. The biosynthesis of methionine is therefore of vital importance to microbial growth and it is therefore an attractive target. Moreover, most of the steps in the methionine biosynthesis pathway are unique to bacteria and plants; their absence in mammals reduces the probability of unwanted side effects. We have studied one of the enzymes required for methionine biosynthesis, namely, NCBI protein NP_940074, which has been annotated as a pyridoxal-5'-phosphate (PLP-)dependent C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria. The threat of diphtheria, even in countries with good coverage in childhood immunization programs by vaccination, has not disappeared and outbreaks in many European countries have revealed a phenomenon of decreasing immunity to diphtheria among adults [Galazka, 2000]. The treatment of diphtheria patients is based on administration of antibiotics to eliminate the corynebacteria from the site of infection, thus stopping ongoing toxin production with or without additional treatment with specific immunoglobulins [Wagner et al., 2009]. Thus, while diphtheria antitoxin (DAT) contributes to reducing the case fatality rates of infections due to this microorganism, it is unlikely, in the foreseeable future, that it will substantially reduce the world’s consumption of antimicrobial agents. This highlights the importance of the development of new antibacterial drugs. C-S lyase catalyzes the ,-elimination of sulfur-containing amino acids, such as L-cystathionine (L-Cth), to generate ammonia, pyruvate, and homocysteine, the penultimate step in methionine biosynthesis transsulfuration pathway. The crystal structure of C. diphtheriae C-S lyase has been determined by X-ray crystallography at 1.99 Å resolution (PDB code: 3FDB, Joint Center for Structural Genomics, http://www.jcsg.org/). According to its folding pattern, the enzyme belongs to the fold type I family of the PLP-dependent enzymes [Schneider et al., 2000]. The commonly known fold type I PLP-dependent enzymes have an aromatic amino acid residue located at the re face of the PLP-Lys internal aldimine and stacking with the pyridine ring of PLP. The structure shows that a conserved binding site for PLP which is covalently linked to Lys222 has a stacking interaction with Tyr114 and H-bond interaction with Asn160, Asp188, and His191. There is one molecule in each asymmetric unit (PDB code: 3FDB). Although the crystal structure has been solved, no protein characterization and structure-based design studies have been reported to date. We overexpressed the protein in E. coli and the availability of the protein in purified form has allowed us to obtain an insight into the biochemical properties of the enzyme by thorough characterization of its kinetic and spectral properties. Substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein have been analyzed [Astegno et al., 2013]. Structural comparison of the enzyme with cystalysin from Treponema denticola indicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C. diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics.