Showing papers on "Ribostamycin published in 1999"

Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry

Abstract: We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS–MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409–G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule/RNA interactions useful in the design of selective new antimicrobial drugs.

Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry (16S RNAy18S RNAyFourier transform ion cyclotron resonance MSyaminoglycoside)

Abstract: We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of amino- glycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycosideyRNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apra- mycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS-MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409-G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small moleculeyRNA interactions useful in the design of selective new antimicrobial drugs. The aminoglycoside antibiotics are potent inhibitors of protein synthesis and RNA splicing in vitro and in vivo (1-6). As the first class of compounds known to bind specifically to subdo- mains of larger RNA sequences, they are useful for under- standing the design principles required to produce new classes of therapeutic agents to bind RNAs (7, 8). The best charac- terized aminoglycoside binding site is the decoding region of the small ribosomal subunit. The structures of paromomycin and gentamicin C1a complexes with a model RNA subdomain have been determined by using NMR (9-12). These amino- glycosides make hydrogen-bonding, electrostatic, and hydro- phobic contacts that contribute to their high binding affinity for the RNA target. We have developed methods that facilitate quantitative analysis of the binding characteristics of multiple aminoglyco- sides with a series of structural variants of the RNA to decipher further the molecular details of how different classes of aminoglycosides can discriminate among structurally related RNAs. We show that electrospray ionization MS (ESI-MS) has unique advantages for rapid characterization of RNA binding with aminoglycosides or any chemical or biological substrate. Mixtures of compounds can be screened in parallel with a high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, because the exact molecular mass of each ligand serves as a unique intrinsic label. Because ESI-MS measurements are performed from solutions of the RNA(s) and small molecules, the relative concentrations of the components and buffers can be adjusted to determine binding constants over a broad range of conditions (13-15). The location of binding has been ascertained with ESI-MSyMS protection experiments performed selectively on ions from the free RNA and the complex (16, 17). We also demonstrate that several RNA targets can be screened simultaneously against mixtures of molecules (18).

Molecular cloning of the gene for the key carbocycle-forming enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics and its comparison with dehydroquinate synthase.

Abstract: The 2-deoxystreptamine aglycon is a common structural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin. A key enzyme involved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-scyllo-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. The recent success of isolating the 2-deoxy-scyllo-inosose synthase from Bacillus circulans prompted us to clone the gene responsible for this important enzyme by the use of reverse genetics approach. With the aid of DNA probes constructed on the basis of the amino-terminal sequence of the purified 42 kDa subunit of the enzyme, the responsible gene btrC was successfully cloned. Subsequently the btrC gene was heterologously expressed in Escherichia coli, and the 2-deoxy-scyllo-inosose synthase activity of the recombinant polypeptide was confirmed by chemical analysis. The btrC gene encodes a protein composed of 368 amino acids with a molecular mass of 40.7 kDa. Our previous proposal for the similarity of 2-deoxy-scyllo-inosose synthase to dehydroquinate synthase has been confirmed on the basis of their amino acid sequences. Significant differences in the sequences can also be observed however, particularly in the crucial substrate recognition regions. Comparison of the BtrC sequence with those of biosynthetic enzymes for other related microbial products is also discussed.