| SynercidRTM is used to treat infections caused by vancomycin resistant Enterococcus faecium, methicillin resistant Staphylococcus species and some Streptococcus species. It belongs to a class of antimicrobials, known as streptogramins, which is comprised of Group A (e.g. dalfopristin) and Group B (e.g. quinupristin) compounds. Independently, these compounds are bacteriostatic against Gram-positive bacteria, however, in combination they exhibit synergistic bactericidal effects due to permanent inhibition of the bacterial 50S ribosomal subunit. Resistance to the Group B streptogramins is conferred enzymatically by streptogramin B lyases (Vgb), which cleave the ring structure of these peptide drugs.;A comparison of Vgb and the ribosome structures reveals that Mg 2+ facilitates the binding of the antibiotic through the 3-hydroxypicolinic acid moiety in both targets, however, the predominant associations are due to van der Waals interactions. In Vgb, interactions between the enzyme and the substrate are predominantly through interactions parallel to the plane of the lactone, while in the ribosome the antibiotic bound is at the periphery of the ring. This allows for extension of the antibiotic at the L-phenylglycine moiety when bound to the ribosome, while such an alteration would preclude efficient binding to Vgb. Lastly, we have characterized an engineered Group B streptogramin that has a variation at the L-phenylglycine position and we find that this new ligand retains antibacterial properties while it is a much weaker substrate for Vgb. This information has laid out the foundation for further structure-based drug design of Group B streptogramins that will bind the ribosome with greater affinity and not be susceptible to inactivation by Vgb.;Here we present the first crystal structure of a streptogramin B lyase from Staphylococcus aureus to a resolution of 1.65 A. High-resolution data sets were collected for both the native and L-selenomethionine substituted Vgb and the structure was solved using a combination of MAD and molecular replacement techniques. The structure of Vgb, is that of a beta-propeller consisting of seven blades made of highly twisted beta-sheets arranged in a circular array. A similar fold has been observed in the WD40, Kelch, YWTD, PQQ and AxSPD families, however, Vgb has no sequence similarity to these proteins. This identifies Vgb as possessing a novel beta- propeller motif. Initial attempts to crystallize Vgb with a catalytically inactive mutant were met with limited success due to problems with crystal packing. Using structure-inspired loop mutagenesis, we were able to crystallize Vgb in a new crystal form conducive to substrate binding. This structure reveals that the antibiotic, quinupristin, binds in a large depression on the top face of Vgb. Guided by the structure, we propose a reaction mechanism in which the initial proton abstraction is facilitated by a Mg2+ linked conjugated system, with His270, His228, Try18, Glu268, and Glu284 playing roles in the catalytic mechanism. Since Mg2+ is only indirectly involved in the opening of the lactone ring, this represents a novel enzymatic mechanism. |
