-
Chemistry & Biology Apr 2004The Salmonella enterica chromosomally encoded AAC(6')-Iy has been shown to confer broad aminoglycoside resistance in strains in which the structural gene is expressed....
The Salmonella enterica chromosomally encoded AAC(6')-Iy has been shown to confer broad aminoglycoside resistance in strains in which the structural gene is expressed. The three-dimensional structures reported place the enzyme in the large Gcn5-related N-acetyltransferase (GNAT) superfamily. The structure of the CoA-ribostamycin ternary complex allows us to propose a chemical mechanism for the reaction, and comparison with the Mycobacterium tuberculosis AAC(2')-CoA-ribostamycin complex allows us to define how regioselectivity of acetylation is achieved. The AAC(6')-Iy dimer is most structurally similar to the Saccharomyces cerevisiae Hpa2-encoded histone acetyltransferase. We demonstrate that AAC(6')-Iy catalyzes both acetyl-CoA-dependent self-alpha-N-acetylation and acetylation of eukaryotic histone proteins and the human histone H3 N-terminal peptide. These structural and catalytic similarities lead us to propose that chromosomally encoded bacterial acetyltransferases, including those functionally identified as aminoglycoside acetyltransferases, are the evolutionary progenitors of the eukaryotic histone acetyltransferases.
Topics: Acetylation; Acetyltransferases; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Catalysis; Crystallography, X-Ray; Dimerization; Histone Acetyltransferases; Histones; Humans; Models, Biological; Models, Molecular; Mycobacterium tuberculosis; Protein Conformation; Protein Structure, Tertiary; Saccharomyces cerevisiae; Salmonella enterica; Structure-Activity Relationship
PubMed: 15123251
DOI: 10.1016/j.chembiol.2004.03.017 -
The Journal of Antimicrobial... Dec 1987An aminoglycoside-acetylating enzyme produced by a strain of Escherichia coli with an unusual resistance phenotype was characterized. This enzyme was found to...
An aminoglycoside-acetylating enzyme produced by a strain of Escherichia coli with an unusual resistance phenotype was characterized. This enzyme was found to mono-acetylate apramycin, butirosin, lividomycin and paromomycin and diacetylate ribostamycin and neomycin to give reaction products which were distinguishable by HPLC analysis from those of AAC(2'), AAC(3) and AAC(6') enzymes. The enzyme, however, was not found to acetylate amikacin, fortimicin, geneticin, gentamicin, kanamycin A, netilmicin or tobramycin. The reaction product from the action of this enzyme on apramycin was purified and identified by nuclear magnetic resonance studies as 1-N-acetyl apramycin. The second site at which ribostamycin and neomycin B were modified by this enzyme was not determined but is postulated as the 6'-amino group. It is proposed that this enzyme be named AAC(1).
Topics: Acetyltransferases; Anti-Bacterial Agents; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Drug Resistance, Microbial; Escherichia coli; Magnetic Resonance Spectroscopy; Nebramycin
PubMed: 3326872
DOI: 10.1093/jac/20.6.803 -
Methods in Enzymology 2015With increasing recognition of the roles RNA molecules and RNA/protein complexes play in an unexpected variety of biological processes, understanding of RNA...
With increasing recognition of the roles RNA molecules and RNA/protein complexes play in an unexpected variety of biological processes, understanding of RNA structure-function relationships is of high current importance. To make clean biological interpretations from three-dimensional structures, it is imperative to have high-quality, accurate RNA crystal structures available, and the community has thoroughly embraced that goal. However, due to the many degrees of freedom inherent in RNA structure (especially for the backbone), it is a significant challenge to succeed in building accurate experimental models for RNA structures. This chapter describes the tools and techniques our research group and our collaborators have developed over the years to help RNA structural biologists both evaluate and achieve better accuracy. Expert analysis of large, high-resolution, quality-conscious RNA datasets provides the fundamental information that enables automated methods for robust and efficient error diagnosis in validating RNA structures at all resolutions. The even more crucial goal of correcting the diagnosed outliers has steadily developed toward highly effective, computationally based techniques. Automation enables solving complex issues in large RNA structures, but cannot circumvent the need for thoughtful examination of local details, and so we also provide some guidance for interpreting and acting on the results of current structure validation for RNA.
Topics: Computational Biology; Crystallization; Crystallography, X-Ray; Data Interpretation, Statistical; Humans; Hydrogen Bonding; Models, Molecular; Nucleic Acid Conformation; RNA; RNA Folding; Ribostamycin; Software
PubMed: 26068742
DOI: 10.1016/bs.mie.2015.01.007 -
The Journal of Antimicrobial... Aug 1986Campylobacter coli strain 981 of animal origin was resistant to erythromycin, tetracycline, streptomycin, kanamycin, ribostamycin, neomycin, paromomycin, lividomycin,...
Campylobacter coli strain 981 of animal origin was resistant to erythromycin, tetracycline, streptomycin, kanamycin, ribostamycin, neomycin, paromomycin, lividomycin, and butirosin. Resistance to aminoglycosides of strain 981 was mediated by phosphotransferases APH (3') type-IV and APH (3"). C. coli 981 harboured three plasmids of 24, 34, and 40 Megadaltons respectively. None of these plasmids were transferable to Escherichia coli K-12 by conjugation.
Topics: Aminoglycosides; Anti-Bacterial Agents; Campylobacter; Conjugation, Genetic; DNA, Bacterial; Drug Resistance, Microbial; Escherichia coli; Kanamycin Kinase; Phosphotransferases; Plasmids
PubMed: 3019983
DOI: 10.1093/jac/18.2.153 -
Journal of Molecular Biology Mar 1998Aminoglycoside antibiotics that bind to ribosomal RNA in the aminoacyl-tRNA site (A-site) cause misreading of the genetic code and inhibit translocation. We have...
Aminoglycoside antibiotics that bind to ribosomal RNA in the aminoacyl-tRNA site (A-site) cause misreading of the genetic code and inhibit translocation. We have recently solved the structure of an A-site RNA-paromomycin complex. The structure suggested that rings I and II, common to all aminoglycosides that bind to the A-site, are the minimum motif for specific ribosome binding to affect translation. This hypothesis was tested biochemically and with a detailed comparative NMR study of interaction of the aminoglycosides paromomycin, neomycin, ribostamycin, and neamine with the A-site RNA. Our NMR data show that rings I and II of neomycin-class aminoglycosides are sufficient to confer specificity to the binding of the antibiotics to the model A-site RNA. Neomycin, paromomycin, ribostamycin and neamine bind in the major groove of the A-site RNA in a unique binding pocket formed by non-canonical base pairs and a bulged nucleotide. Similar NMR properties of the RNA and the diverse antibiotics within the different complexes formed with neomycin, paromomycin, ribostamycin and neamine suggest similar structures for these complexes.
Topics: Anti-Bacterial Agents; Binding Sites; Carbohydrate Sequence; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Neomycin; RNA, Ribosomal, 16S
PubMed: 9514735
DOI: 10.1006/jmbi.1997.1552 -
Zhongguo Yao Li Xue Bao = Acta... Jan 1986
Topics: Animals; Anti-Bacterial Agents; Cats; Diaphragm; Female; In Vitro Techniques; Male; Membrane Potentials; Mice; Muscle Contraction; Neuromuscular Junction; Rats; Ribostamycin; Synaptic Transmission; Vas Deferens
PubMed: 3020871
DOI: No ID Found -
Nature Structural Biology Sep 2002AAC(2')-Ic catalyzes the coenzyme A (CoA)-dependent acetylation of the 2' hydroxyl or amino group of a broad spectrum of aminoglycosides. The crystal structure of the...
AAC(2')-Ic catalyzes the coenzyme A (CoA)-dependent acetylation of the 2' hydroxyl or amino group of a broad spectrum of aminoglycosides. The crystal structure of the AAC(2')-Ic from Mycobacterium tuberculosis has been determined in the apo enzyme form and in ternary complexes with CoA and either tobramycin, kanamycin A or ribostamycin, representing the first structures of an aminoglycoside acetyltransferase bound to a drug. The overall fold of AAC(2')-Ic places it in the GCN5-related N-acetyltransferase (GNAT) superfamily. Although the physiological function of AAC(2')-Ic is uncertain, a structural analysis of these high-affinity aminoglycoside complexes suggests that the enzyme may acetylate a key biosynthetic intermediate of mycothiol, the major reducing agent in mycobacteria, and participate in the regulation of cellular redox potential.
Topics: Acetyltransferases; Aminoglycosides; Carbohydrate Sequence; Coenzyme A; Crystallography, X-Ray; Models, Molecular; Mycobacterium tuberculosis; Protein Conformation; Substrate Specificity
PubMed: 12161746
DOI: 10.1038/nsb830 -
Chemistry & Biology Apr 2007Butirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic...
Butirosin, an aminoglycoside antibiotic produced by Bacillus circulans, bears the unique (S)-4-amino-2-hydroxybutyrate (AHBA) side chain, which protects the antibiotic from several common resistance mechanisms. The AHBA side chain is advantageously incorporated into clinically valuable antibiotics such as amikacin and arbekacin by synthetic methods. Therefore, it is of significant interest to explore the biosynthetic origins of this useful moiety. We report here that the AHBA side chain of butirosin is transferred from the acyl carrier protein (ACP) BtrI to the parent aminoglycoside ribostamycin as a gamma-glutamylated dipeptide by the ACP:aminoglycoside acyltransferase BtrH. The protective gamma-glutamyl group is then cleaved by BtrG via an uncommon gamma-glutamyl cyclotransferase mechanism. The application of this pathway to the in vitro enzymatic production of novel AHBA-bearing aminoglycosides is explored with encouraging implications for the preparation of unnatural antibiotics via directed biosynthesis.
Topics: Acyl Carrier Protein; Amino Acids; Aminoglycosides; Bacillus; Bacterial Proteins; Butirosin Sulfate; Recombinant Proteins
PubMed: 17462573
DOI: 10.1016/j.chembiol.2007.02.005 -
Analytical Biochemistry May 1988In order to make accurate kinetic measurements for the substrates of aminoglycoside (AG) phosphotransferases (APHs), we have developed an assay which overcomes many of...
In order to make accurate kinetic measurements for the substrates of aminoglycoside (AG) phosphotransferases (APHs), we have developed an assay which overcomes many of the limitations of currently used assays. We have adapted the coupled spectrophotometric assay (P. R. Goldman and D. B. Northrop (1976) Biochem. Biophys. Res. Commun. 68, 230-236) for use in a spectrofluorometer. At an excitation wavelength of 340 nm, NADH will emit an intensity peak at 450 nm; NAD does not emit under these conditions. Our assay can accurately measure differences of 0.25 microM. For the APH(3')-II encoded on Tn5, we have redetermined the Km's for the AGs, amikacin (AK), kanamycin (KM), and ribostamycin (Rib), and for ATP. Our values for AK (76 microM) were lower than those derived from the spectrophotometric assay; for KM and Rib we obtained Km values of 5.1 and 3.6 microM, respectively. These values were well below the limit of accuracy (10 microM) for the spectrophotometric assay. In addition, we have begun characterization of an APH from a clinical isolate with a low Km for AK. Thus far, we have determined that this enzyme has Km's of approximately 1 microM for both AK and KM. These results show that the assay is well suited for accurate determinations of kinetic constants for low Km substrates of APH enzymes.
Topics: Drug Resistance, Microbial; Escherichia coli; Kanamycin Kinase; Phosphotransferases; Plasmids; Pseudomonas aeruginosa; Spectrometry, Fluorescence
PubMed: 2841886
DOI: 10.1016/0003-2697(88)90135-2 -
Frontiers in Microbiology 2021Multidrug-resistant bacteria from different sources have been steadily emerging, and an increasing number of resistance mechanisms are being uncovered. In this work, we...
Multidrug-resistant bacteria from different sources have been steadily emerging, and an increasing number of resistance mechanisms are being uncovered. In this work, we characterized a novel resistance gene named from an isolate of a novel species, R33 (CCTCC AB 2021339). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 2'--acetyltransferase. Whole-genome sequencing and comparative genomic analysis were performed to elucidate the molecular characteristics of the genome and the genetic context of the resistance gene-related sequences. Among the functionally characterized resistance genes, AAC(2')-If shares the highest amino acid sequence identity of 70.79% with AAC(2')-Ia. AAC(2')-If confers resistance to several aminoglycoside antibiotics, showing the highest resistance activity against ribostamycin and neomycin. The recombinant strain harboring (pUCP20-/DH5α) showed 256- and 128-fold increases in the minimum inhibitory concentration (MIC) levels to ribostamycin and neomycin, respectively, compared with those of the control strains (DH5α and pUCP20/DH5α). The results of the kinetic analysis of AAC(2')-If were consistent with the MIC results of the cloned with the highest catalytic efficiency for ribostamycin ( ratio = [3.72 ± 0.52] × 10 M s). Whole-genome sequencing demonstrated that the gene was located on the chromosome with a relatively unique genetic environment. Identification of a novel aminoglycoside resistance gene in a strain of a novel species will help us find ways to elucidate the complexity of resistance mechanisms in the microbial population.
PubMed: 34867838
DOI: 10.3389/fmicb.2021.711037