Sparsomycin–Linezolid Conjugates Can Promote Ribosomal Translocation

Abstract

Sparsomycin is an antibiotic that targets the peptidyl transferase center of the ribosome and has the ability to promote ribosomal translocation in the absence of EF-G and GTP. Here we show that changes in the configurations at the two chiral centers of sparsomycin, especially at the chiral carbon, can greatly affect its capability to promote ribosomal translocation. More importantly, the incorporation of the pseudo-uracil moiety of sparsomycin into linezolid through a covalent linkage conferred on linezolid derivatives the ability to promote translocation, thus indicating the importance of interactions between this pseudo-uracil moiety, rRNA, and tRNA for promoting translocation. In addition, these translocation promoters can also effectively inhibit spontaneous reverse translocation; this suggests that they might promote forward translocation by trapping the ribosome in the post-translocation state and shifting the equilibrium between the pre- and post-translocation ribosome in the forward direction.

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

“…3). Furthermore, isomerization of the sulfurrich tail of sparsomycin, which interacts with the 50S A site, was recently shown to significantly decrease the ability of sparsomycin to induce translocation (Li et al 2011). Thus, our results support the idea that the ribosome is a Brownian ratchet machine whose spontaneous fluctuation can be rectified into translocation by ligand binding.…”

“…2). No translocation was observed in the presence of linezolid, another A-sitebinding antibiotic (Li et al 2011). There may be several factors that contribute to the decreased translocation potency of other A-site antibiotics in comparison to sparsomycin.…”

“…Furthermore, the translocation efficiency of A-site antibiotics appears to correlate loosely with their affinity for the ribosome, decreasing from sparsomycin (K d < 1 µM) (Lazaro et al 1987) to chloramphenicol (K d ∼ 2-6 µM) (Contreras and Vazquez 1977) and the lincosamides (K d ∼ 4-6 µM) (Contreras and Vazquez 1977;Douthwaite 1992) to linezolid (K d ∼ 20 µM) (Lin et al 1997) and puromycin (K d ∼ 3-4 mM) (Wohlgemuth et al 2006). Recently, it has been demonstrated that while neither linezolid nor the pseudouridine moiety of sparsomycin, which binds to the A and P sites of large subunit, respectively, can stimulate mRNA translocation by themselves, a linezolid-pseudouridine conjugate can induce translocation in a fraction of ribosomes (Li et al 2011). Conjugation of linezolid with the pseudouridine moiety of sparsomycin likely enhances the translocation potency of linezolid by increasing its binding affinity for the ribosome.…”

Structural basis of translation inhibition by cadazolid, a novel quinoxolidinone antibiotic

“…Among the antibiotics that interfere with the peptidyl transferase reaction, oxazolidinones have been in the focus of antibacterial discovery research for many years, and two drugs have been approved for the treatment of infections due to susceptible Gram-positive pathogens 7,8 . Linezolid (LZD) was the first clinically used antibiotic of this class, but a wide variety of oxazolidinone analogues were designed in recent years to improve efficiency against resistant strains 9,10 .…”

Sparsomycin Biosynthesis Highlights Unusual Module Architecture and Processing Mechanism in Non-ribosomal Peptide Synthetase

“…The addition of SpsM, but not any other reductases, to the SpsQ/R reaction system yielded a new compound 2, which was shown to be a dipeptidyl alcohol that had previously been reported (Figure 3b and S7). 18 No dipeptidyl aldehyde was detected by extracted mass analysis. In addition, disruption of spsM in S. sparsogenes completely abolished the production of sparsomycin (Figure 2a), confirming the critical function of the encoding enzyme.…”