The interaction of rhodium(II) carboxylates with enzymes.
The effect of rhodium(II) acetate, propionate, and methoxyacetate on the activity of 17 enzymes was evaluated. The enzymes were preincubated with the rhodium(II) complexes in order to detect irreversible inhibition. All enzymes that have essential sulfhydryl groups in or near their active site were found to be irreversibly inhibited. Those enzymes without essential sulfhydryl groups were not affected. In each case, the rate of inactivation closely paralleled the observed toxicity and antitumor activity of rhodium(II) carboxylates; that is, rhodium(II) propionate greater than rhodium(II) acetate greater than rhodium(II) methoxyacetate. In addition, those enzymes that have been demonstrated to be most sensitive to established sulfhydryl inhibitors, such as glyceraldehyde-3-phosphate dehydrogenase, were also most sensitive to rhodium(II) carboxylate inactivation. Proton nuclear magnetic resonance measurements made during the titration of rhodium(II) acetate with cysteine showed that breakdown of the carboxylate cage occurred as a result of reaction with this sulfhydryl-containing amino acid. (+info)
Prodigious substrate specificity of AAC(6')-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci.
BACKGROUND: High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6')-APH(2"), an enzyme with 6'-N-acetyltransferase and 2"-O-phosphotransferase activities. The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents. RESULTS: In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6')-APH(2") in Bacillus subtilis. The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities. Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6')-APH(2")'s dramatic negative impact on clinical aminoglycoside therapy. Both lividomycin A and paromomycin, aminoglycosides lacking a 6'-amino group, were acetylated by AAC(6')-APH(2"). The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation. Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3'-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3'-OH and the 3"'-OH of ring D, both unprecedented observations for this enzyme. Furthermore, the phosphorylation site of lividomycin A was determined to be the 5"-OH of the pentose ring C. CONCLUSIONS: The bifunctional AAC(6')-APH(2") has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups. The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design. (+info)
Single atom modification (O-->S) of tRNA confers ribosome binding.
Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM). We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding. The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM). Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA. The unmodified ASLLys3UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA. (+info)
Molecular dynamics studies of U1A-RNA complexes.
The U1A protein binds to a hairpin RNA and an internal-loop RNA with picomolar affinities. To probe the molecular basis of U1A binding, we performed state-of-the-art nanosecond molecular dynamics simulations on both complexes. The good agreement with experimental structures supports the protocols used in the simulations. We compare the dynamics, hydrogen-bonding occupancies, and interfacial flexibility of both complexes and also describe a rigid-body motion in the U1A-internal loop complex that is not observed in the U1A-hairpin simulation. We relate these observations to experimental mutational studies and highlight their significance in U1A binding affinity and specificity. (+info)
An examination of coaxial stacking of helical stems in a pseudoknot motif: the gene 32 messenger RNA pseudoknot of bacteriophage T2.
The RNA pseudoknot located at the 5' end of the gene 32 messenger RNA of bacteriophage T2 contains two A-form helical stems connected by two loops, in an H-type pseudoknot topology. A combination of multidimensional NMR methods and isotope labeling were used to investigate the pseudoknot structure, resulting in a more detailed structural model than provided by earlier homonuclear NMR studies. Of particular significance, the interface between the stacked helical stems within the pseudoknot motif is described in detail. The two stems are stacked in a coaxial manner, with an approximately 18 degrees rotation of stem1 relative to stem2 about an axis that is parallel to the helical axis. This rotation serves to relieve what would otherwise be a relatively close phosphate-phosphate contact at the junction of the two stems, while preserving the stabilizing effects of base stacking. The ability of the NMR data to determine pseudoknot bending was critically assessed. The data were found to be a modestly precise indicator of pseudoknot bending, with the angle between the helical axes of stem1 and stem2 being in the range of 15+/-15 degrees. Pseudoknot models with bend angles within this range are equally consistent with the data, since they differ by only small amounts in the relatively short-range interproton distances from which the structure was derived. The gene 32 messenger RNA pseudoknot was compared with other RNA structures with coaxial or near-coaxial stacked helical stems. (+info)
Purinogen is not an endogenous substrate used in endothelial cells during substrate deprivation.
Porcine aortic endothelial cells (PAEC) are known to be metabolically robust. They are capable of surviving extended periods of complete lack of exogenous substrate, and purine release has been shown to be significantly up-regulated. The endogenous substrates used during substrate deprivation, as well as the sources responsible for the increased purine release, have not been completely identified. We tested the possibility that a phosphoglyceroyl-ATP-containing polymer, purinogen, might support PAEC hibernation induced by lack of exogenous substrate. This involved isolation of the acid-insoluble fraction of PAEC, which was presumed to contain purinogen, and analysis by HPLC and 31P NMR. No evidence supporting the presence of triphosphate-containing compounds (purinogen) was found. Similar results were obtained in the rat heart. The majority of the products in the acid-insoluble, alkaline-treated fraction were identified as RNA degradation products (2'- and 3'-nucleoside monophosphates). A [14C]adenosine labelling experiment showed that incorporation of adenosine into the acid-insoluble fraction was almost completely prevented after inhibition of RNA synthesis with actinomycin D. Furthermore, RNA isolated from PAEC and subsequently treated with alkali showed a profile that was almost identical with the HPLC profile of the acid-insoluble fraction. Finally, substrate-free incubation of the cells did not quantitatively or qualitatively influence the distribution of acid-insoluble derivatives. We conclude that PAEC survival during the absence of exogenous substrate is not supported by purinogen but rather by some other, yet-to-be-identified, endogenous substrate. (+info)
Accumulation of astaxanthin all-E, 9Z and 13Z geometrical isomers and 3 and 3' RS optical isomers in rainbow trout (Oncorhynchus mykiss) is selective.
Concentrations of all-E-, 9Z- and 13Z- geometrical and (3R,3'R), (3R, 3'S) and (3S,3'S) optical isomers of astaxanthin were determined in rainbow trout liver, gut tissues, kidney, skin and blood plasma to evaluate their body distribution. Two cold-pelleted diets containing predominantly all-E-astaxanthin (36.9 mg/kg astaxanthin, 97% all-E-, 0.4% 9Z-, 1.5% 13Z-astaxanthin, and 1.1% other isomers, respectively) or a mixture of all-E- and Z-astaxanthins (35.4 mg/kg astaxanthin, 64% all-E-, 18.7% 9Z-, 12.3% 13Z-astaxanthin, and 2.0% other isomers, respectively), were fed to duplicate groups of trout for 69 d. Individual E/Z isomers were identified by VIS- and 1H-NMR-spectrometry, and quantified by high-performance liquid chromatography. Significantly higher total carotenoid concentration was observed in plasma of trout fed diets with all-E-astaxanthin (P < 0.05). The relative E/Z-isomer concentrations of plasma, skin and kidney were not significantly different among groups, whereas all-E-astaxanthin was higher in intestinal tissues and 13Z-astaxanthin was lower in liver of trout fed all-E-astaxanthin (P < 0.05). The relative amount of hepatic 13Z-astaxanthin (39-49% of total astaxanthin) was higher than in all other samples (P < 0.05). Synthetic, optically inactive astaxanthin was used in all experiments, and the determined dietary ratio between the 3R,3'R:3R, 3'S (meso):3S,3'S optical isomers was 25.3:49.6:25.1. The distribution of R/S-astaxanthin isomers in feces, blood, liver and fillet was similar to that in the diets. The ratio between (3S,3'S)- and (3R,3'R)-astaxanthin in the skin and posterior kidney was ca. 2:1 and 3:1, respectively, regardless of dietary E/Z-astaxanthin composition. The results show that geometrical and optical isomers of astaxanthin are distributed selectively in different tissues of rainbow trout. (+info)
Biophysical characterization of the structure of the amino-terminal region of gp41 of HIV-1. Implications on viral fusion mechanism.
A peptide of 51 amino acids corresponding to the NH2-terminal region (5-55) of the glycoprotein gp41 of human immunodeficiency virus type 1 was synthesized to study its conformation and assembly. Nuclear magnetic resonance experiments indicated the sequence NH2-terminal to the leucine zipper-like domain of gp41 was induced into helix in the micellar solution, in agreement with circular dichroism data. Light scattering experiment showed that the peptide molecules self-assembled in water into trimeric structure on average. That the peptide molecules oligomerize in aqueous solution was supported by gel filtration and diffusion coefficient experiments. Molecular dynamics simulation based on the NMR data revealed a flexible region adjacent to the hydrophobic NH2 terminus of gp41. The biological significance of the present findings on the conformational flexibility and the propensity of oligomerization of the peptide may be envisioned by a proposed model for the interaction of gp41 with membranes during fusion process. (+info)