DNA Gyrase as a Target for Quinolones.

Bacterial DNA gyrase is a type II topoisomerase that can introduce negative supercoils to DNA substrates and is a clinically-relevant target for the development of new antibacterials. DNA gyrase is one of the primary targets of quinolones, broad-spectrum antibacterial agents and are used as a first-line drug for various types of infections. However, currently used quinolones are becoming less effective due to drug resistance. Common resistance comes in the form of mutation in enzyme targets, with this type being the most clinically relevant. Additional mechanisms, conducive to quinolone resistance, are arbitrated by chromosomal mutations and/or plasmid-gene uptake that can alter quinolone cellular concentration and interaction with the target, or affect drug metabolism. Significant synthetic strategies have been employed to modify the quinolone scaffold and/or develop novel quinolones to overcome the resistance problem. This review discusses the development of quinolone antibiotics targeting DNA gyrase to overcome bacterial resistance and reduce toxicity. Moreover, structural activity relationship (SAR) data included in this review could be useful for the development of future generations of quinolone antibiotics.

A counterintuitive Mg2+-dependent and modification-assisted functional folding of mitochondrial tRNAs.

Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg2+ is critical for folding of the truncated D-domain of bovine mtRNAMet with the tRNA's T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg2+. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg2+ prevents misfolding of the 5'-half of bovine mtRNAMet facilitating its correct interaction with the T-domain. The interaction of the mtRNAMet D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (Kd=25.3, 24.6 and 44.4 microM, respectively). Mg2+ also affected the folding interaction of a yeast mtRNALeu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNALeu. The D-domain modification, dihydrouridine, facilitated mtRNALeu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.

The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit.

Bovine mitochondrial translational initiation factor 2 (IF-2(mt)) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III-VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2(mt) were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2(mt) was strong (K(d) approximately 10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2(mt) to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2(mt) can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2(mt) variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2(mt). IF-2(mt) contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.

Interaction of mitochondrial initiation factor 2 with mitochondrial fMet-tRNA.

The mammalian mitochondrial genome contains a single tRNA(Met) gene that gives rise to the initiator and elongator tRNA(Met). It is generally believed that mitochondrial protein synthesis begins with formylmethionyl-tRNA, which indicates that the formylation of mitochondrial Met-tRNA specifies its participation in initiation through its interaction with initiation factor 2 (IF-2). However, recent studies in yeast mitochondria, suggest that formylation is not required for protein synthesis. In addition, bovine IF-2(mt) could replace yeast IF-2(mt) in strains that lack fMet-tRNA which suggests that this paradigm may extend to mammalian mitochondria. Here, the importance of the formylation of mitochondrial Met-tRNA for the interaction with IF-2(mt) was investigated by measuring the ability of bovine IF-2(mt) to bind mitochondrial fMet-tRNA. In direct binding experiments, bovine IF-2(mt) has a 25-fold greater affinity for mitochondrial fMet-tRNA than Met-tRNA, using either the native mitochondrial tRNA(Met) or an in vitro transcript of bovine mitochondrial tRNA(Met). In addition, IF-2(mt) will not effectively stimulate mitochondrial Met-tRNA binding to mitochondrial ribosomes, exhibiting a 50-fold preference for fMet-tRNA over Met-tRNA in this assay. Finally, the region of IF-2(mt) responsible for the interaction with fMet-tRNA was mapped to the C2 sub-domain of domain VI of this factor.

Characterization of the human mitochondrial methionyl-tRNA synthetase.

Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database. The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence. Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn(2+) binding motif and C-terminal dimerization region found in MetRSs from various organisms. The mature form of human mtMetRS has been cloned and expressed in Escherichia coli. Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa. The kinetic parameters for activation of methionine have been determined for the purified enzyme. The K(M) and k(cat) for aminoacylation of E. coli initiator tRNA(f)(Met) are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA(Met) (mtRNA(Met)) have been determined. To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA(Met) and of an in vitro transcript of the bovine mtRNA(Met) have also been investigated.