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

Sparsomycin is a model protein synthesis inhibitor that blocks peptide bond formation by binding to the large ribosome subunit. It is a unique dipeptidyl alcohol, consisting of a uracil acrylic acid moiety and a monooxo-dithioacetal group. To elucidate the biosynthetic logic of sparsomycin, a biosynthetic gene cluster for sparsomycin was identified from the producer Streptomyces sparsogenes by genome mining, targeted gene mutations, and heterologous expression. Both the genetic and enzymatic studies revealed a minimum set of non-ribosomal peptide synthetases needed for generating the dipeptidyl alcohol scaffold of sparsomycin, featuring unusual mechanisms in dipeptidyl assembly and off-loading.

Sparsomycin-linezolid conjugates can promote ribosomal translocation.

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.

Calculation of the standard binding free energy of sparsomycin to the ribosomal peptidyl-transferase P-site using molecular dynamics simulations with restraining potentials.

The standard (absolute) binding free energy of the antibiotic sparsomycin with the 50S bacteria ribosomal subunit is calculated using molecular dynamics (MD) free energy perturbation (FEP) simulations with restraining potentials developed by Wang et al. [Biophys. J. 91:2798-2814 (2006)]. In the simulation protocol, restraining potentials are activated for the orientational and translational movements of the ligand relative to the binding site when it is decoupled from the binding pocket, and then released once the ligand fully interacts with the rest of the system. A reduced system is simulated to decrease the computational cost of the FEP/MD calculations and the effects of the surrounding atoms are incorporated using the generalized solvent boundary potential (GSBP) method. The loss of conformational freedom of the ligand upon binding is characterized using the potential of mean force (PMF) as a function of the root-mean-square deviation (RMSD) relative to the bound conformation. The number of water molecules in the binding pocket is allowed to fluctuate dynamically in response to the ligand during the calculations by combining FEP/MD with grand canonical Monte Carlo (GCMC) simulations. The calculated binding free energy is about -6 kcal/mol, which is in reasonable agreement with the experimental value. The information gleaned from this study provides new insight on the recognition of ribosome by sparsomycin and highlights the challenges in calculations of absolute binding free energies in these systems.

Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit.

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.

Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.

Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.

Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome.

Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure-function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site.