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1.
mBio ; 12(1)2021 02 09.
Article in English | MEDLINE | ID: mdl-33563840

ABSTRACT

Apramycin is an aminoglycoside antibiotic with the potential to be developed to combat multidrug-resistant pathogens. Its unique structure evades the clinically widespread mechanisms of aminoglycoside resistance that currently compromise the efficacy of other members in this drug class. Of the aminoglycoside-modifying enzymes that chemically alter these antibiotics, only AAC(3)-IVa has been demonstrated to confer resistance to apramycin through N-acetylation. Knowledge of other modification mechanisms is important to successfully develop apramycin for clinical use. Here, we show that ApmA is structurally unique among the previously described aminoglycoside-modifying enzymes and capable of conferring a high level of resistance to apramycin. In vitro experiments indicated ApmA to be an N-acetyltransferase, but in contrast to AAC(3)-IVa, ApmA has a unique regiospecificity of the acetyl transfer to the N2' position of apramycin. Crystallographic analysis of ApmA conclusively showed that this enzyme is an acetyltransferase from the left-handed ß-helix protein superfamily (LßH) with a conserved active site architecture. The success of apramycin will be dependent on consideration of the impact of this potential form of clinical resistance.IMPORTANCE Apramycin is an aminoglycoside antibiotic that has been traditionally used in veterinary medicine. Recently, it has become an attractive candidate to repurpose in the fight against multidrug-resistant pathogens prioritized by the World Health Organization. Its atypical structure circumvents most of the clinically relevant mechanisms of resistance that impact this class of antibiotics. Prior to repurposing apramycin, it is important to understand the resistance mechanisms that could be a liability. Our study characterizes the most recently identified apramycin resistance element, apmA We show ApmA does not belong to the protein families typically associated with aminoglycoside resistance and is responsible for modifying a different site on the molecule. The data presented will be critical in the development of apramycin derivatives that will evade apmA in the event it becomes prevalent in the clinic.


Subject(s)
Acetyltransferases/chemistry , Acetyltransferases/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Escherichia coli/drug effects , Nebramycin/analogs & derivatives , Acetylation , Aminoglycosides/chemistry , Crystallization , Drug Resistance, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Humans , Microbial Sensitivity Tests , Nebramycin/chemistry , Nebramycin/metabolism
2.
J Am Chem Soc ; 138(20): 6427-35, 2016 05 25.
Article in English | MEDLINE | ID: mdl-27120352

ABSTRACT

Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products.


Subject(s)
Nebramycin/analogs & derivatives , Oxygen/chemistry , Aminoglycosides/metabolism , Carbohydrate Sequence , Catalysis , Nebramycin/biosynthesis , Nebramycin/chemistry , Nebramycin/metabolism , Oxidoreductases/metabolism , Substrate Specificity
3.
Biochemistry ; 52(30): 5125-32, 2013 Jul 30.
Article in English | MEDLINE | ID: mdl-23837529

ABSTRACT

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis . Eis from M. tuberculosis (Eis_Mtb) and M. smegmatis (Eis_Msm) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_Mtb is divided into two narrow channels, whereas Eis_Msm contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_Mtb, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_Msm, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_Mtb and for the functional differences from Eis_Msm, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_Mtb by mutating them into their Eis_Msm counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_Mtb. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_Mtb nomenclature) dictate the differences between the acetylation efficiencies of Eis_Mtb and Eis_Msm for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_Mtb into Ala plays a role in APR acetylation.


Subject(s)
Acetyltransferases/metabolism , Aminoglycosides/metabolism , Antibiotics, Antitubercular/metabolism , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/enzymology , Acetylation , Acetyltransferases/chemistry , Acetyltransferases/genetics , Amino Acid Sequence , Amino Acid Substitution , Aminoglycosides/chemistry , Aminoglycosides/pharmacology , Antibiotics, Antitubercular/chemistry , Antibiotics, Antitubercular/pharmacology , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Drug Resistance, Multiple, Bacterial , Kanamycin/chemistry , Kanamycin/metabolism , Kanamycin/pharmacology , Kinetics , Molecular Conformation , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Nebramycin/analogs & derivatives , Nebramycin/chemistry , Nebramycin/metabolism , Nebramycin/pharmacology , Paromomycin/chemistry , Paromomycin/metabolism , Paromomycin/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Substrate Specificity
4.
Proc Natl Acad Sci U S A ; 110(33): 13333-8, 2013 Aug 13.
Article in English | MEDLINE | ID: mdl-23898171

ABSTRACT

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.


Subject(s)
Gentamicins/chemistry , Gentamicins/pharmacology , Leishmania/drug effects , Leishmaniasis/drug therapy , Models, Molecular , Ribosomal Proteins/chemistry , Crystallization , Gentamicins/metabolism , Leishmania/growth & development , Molecular Structure , Nebramycin/analogs & derivatives , Nebramycin/chemistry , Nebramycin/metabolism , Nebramycin/pharmacology , Protein Binding , Protein Conformation , Ribosomal Proteins/metabolism
5.
Dalton Trans ; (7): 1123-30, 2009 Feb 21.
Article in English | MEDLINE | ID: mdl-19322482

ABSTRACT

The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).


Subject(s)
Copper/chemistry , DNA/chemistry , Nebramycin/analogs & derivatives , Oxidative Stress/drug effects , Plasmids/chemistry , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Circular Dichroism , Copper/metabolism , DNA/metabolism , Electron Spin Resonance Spectroscopy , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Models, Chemical , Nebramycin/chemistry , Nebramycin/metabolism , Oxidants/pharmacology , Thermodynamics
6.
Blood Cells Mol Dis ; 38(3): 193-8, 2007.
Article in English | MEDLINE | ID: mdl-17258916

ABSTRACT

Aminoglycoside antibiotics bind specifically to the bacterial ribosomal decoding-site RNA and thereby interfere with fidelity but not efficiency of translation. Apramycin stands out among aminoglycosides for its mechanism of action which is based on blocking translocation and its ability to bind also to the eukaryotic decoding site despite differences in key residues required for apramycin recognition by the bacterial target. To elucidate molecular recognition of the eukaryotic decoding site by apramycin we have determined the crystal structure of an oligoribonucleotide containing the human sequence free and in complex with the antibiotic at 1.5 A resolution. The drug binds in the deep groove of the RNA which forms a continuously stacked helix comprising non-canonical C.A and G.A base pairs and a bulged-out adenine. The binding mode of apramycin at the human decoding-site RNA is distinct from aminoglycoside recognition of the bacterial target, suggesting a molecular basis for the actions of apramycin in eukaryotes and bacteria.


Subject(s)
Models, Molecular , Nebramycin/analogs & derivatives , RNA, Ribosomal , Binding Sites , Humans , Molecular Structure , Nebramycin/chemistry , Nebramycin/metabolism , Nucleic Acid Conformation , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , Structure-Activity Relationship
7.
Biochemistry ; 44(49): 16275-83, 2005 Dec 13.
Article in English | MEDLINE | ID: mdl-16331988

ABSTRACT

The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.


Subject(s)
Acetyltransferases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Acetyltransferases/chemistry , Acetyltransferases/genetics , Anti-Bacterial Agents/metabolism , Calorimetry , Drug Resistance, Bacterial , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Humans , Hydrogen-Ion Concentration , Isotopes/chemistry , Molecular Structure , Nebramycin/analogs & derivatives , Nebramycin/metabolism , Substrate Specificity
8.
Biotechnol Lett ; 27(15): 1129-34, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16132864

ABSTRACT

An ORF located immediately downstream of glnR gene was cloned from Amycolatopsis mediterranei U32 and was named lh3. Sequence analysis revealed that lh3 encodes a putative acetyltransferase, which shows high amino acid sequence similarities to the mycothiol synthase (MshD) from other actinomycetes. For functional analysis, mutation in lh3 gene was generated by gene replacement with an apramycin resistance gene through homologous recombination. Compared with the wild type strain, the resulting mutant was more sensitive to H2O2, apramycin and erythromycin by two- to three-fold. These results suggest that the lh3 gene plays an important role in the course of detoxification in A. mediterranei U32.


Subject(s)
Acetyltransferases/genetics , Actinomycetales/metabolism , Gene Expression Regulation, Fungal , Repressor Proteins/genetics , Rifamycins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Acetyltransferases/metabolism , Amino Acid Sequence , Cloning, Molecular , DNA/metabolism , Erythromycin/pharmacology , Hydrogen Peroxide/pharmacology , Molecular Sequence Data , Mutation , Nebramycin/analogs & derivatives , Nebramycin/metabolism , Open Reading Frames , Phenotype , Physical Chromosome Mapping , Plasmids/metabolism , Protein Structure, Tertiary , Recombination, Genetic , Sequence Homology, Amino Acid
10.
J Am Chem Soc ; 126(47): 15402-4, 2004 Dec 01.
Article in English | MEDLINE | ID: mdl-15563166

ABSTRACT

A major mechanism for bacterial resistance to antibiotics is through the acquisition of a plasmid coding for resistance-mediating proteins. Described herein is a strategy to eliminate these plasmids from bacteria, thus resensitizing the bacteria to antibiotics. This approach involves mimicking a natural mechanism for plasmid elimination, known as plasmid incompatibility. The compound apramycin was identified as a tight binder to SLI RNA (Kd = 93 nM), the in vivo target of the plasmid incompatibility determinate RNA I, and footprinting/mutagenesis studies indicate apramycin binds SLI in the important regulatory region that dictates plasmid replication control and incompatibility. In vivo studies demonstrate that this compound causes significant plasmid loss and resensitizes bacteria to conventional antibiotics. The demonstration that a small molecule can mimic incompatibility, cause plasmid elimination, and resensitize bacteria to antibiotics opens up new targets for antibacterial research.


Subject(s)
Aminoglycosides/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Nebramycin/analogs & derivatives , R Factors/antagonists & inhibitors , R Factors/genetics , RNA, Untranslated/genetics , Aminoglycosides/metabolism , Base Sequence , Biomimetic Materials/metabolism , Biomimetic Materials/pharmacology , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Nebramycin/metabolism , Nebramycin/pharmacology , Nucleic Acid Conformation , R Factors/metabolism , RNA, Messenger/genetics , RNA, Untranslated/metabolism , Trans-Activators/genetics , beta-Lactamases/genetics
11.
J Antimicrob Chemother ; 20(6): 803-13, 1987 Dec.
Article in English | MEDLINE | ID: mdl-3326872

ABSTRACT

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).


Subject(s)
Acetyltransferases/isolation & purification , Anti-Bacterial Agents/metabolism , Escherichia coli/enzymology , Nebramycin/metabolism , Acetyltransferases/metabolism , Anti-Bacterial Agents/pharmacology , Carbon Radioisotopes , Chromatography, High Pressure Liquid , Drug Resistance, Microbial , Escherichia coli/drug effects , Magnetic Resonance Spectroscopy , Nebramycin/analogs & derivatives , Nebramycin/pharmacology
12.
Vet Med Nauki ; 24(1): 64-71, 1987.
Article in Bulgarian | MEDLINE | ID: mdl-3617468

ABSTRACT

The serum concentrations of apramycin in calves were studied after i/v application at the rate of 20 mg/kg body mass, after i/m injection at 20-40 mg/kg b. m., and after oral administration (with milk) at 40 mg/kg b. m. in terms of establishing certain pharmacokinetic parameters. It was found that at i/v route of application the concentrations of the antibiotic ranged above 2 micrograms/cm3 within the interval from the 15th min up to the 8h hour. The time of half-distribution (t1/2 alpha) was 0.28 h, while the biologic half-life of half-elimination (t1/2 beta) was 2.31 h. After muscular application of apramycin at 20 and 40 mg/kg it was rapidly adsorbed at the site of injection; the maximum concentrations were 99 micrograms/cm3 and 202 micrograms/cm3, resp., from the first to the second hour. Levels above 2 micrograms/cm3 were found in the blood serum in the course of 8 to 10 hours when the antibiotic was applied at 20 mg/kg b. m., and they have persisted for more than 12 hours when it was administered at the rate of 40 mg/kg. The biologic half-life (t1/2 beta) was 1.63 h and 1.97 h, respectively. Following the oral administration of the antibiotic at 40 mg/kg subtherapeutic levels were established up to the 24 th hour, with the exception of the interval of the 6 th to 8 th when the concentrations were below the minimum therapeutic one (2 micrograms/cm3).(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Anti-Bacterial Agents/metabolism , Cattle/metabolism , Nebramycin/metabolism , Animals , Body Burden , Female , Kinetics , Male , Meat/analysis , Nebramycin/administration & dosage , Nebramycin/analogs & derivatives , Time Factors , Tissue Distribution
13.
J Vet Pharmacol Ther ; 8(1): 95-104, 1985 Mar.
Article in English | MEDLINE | ID: mdl-3989905

ABSTRACT

The minimal inhibitory concentrations (MIC) of apramycin, a unique aminocyclitol antibiotic, were compared with the MIC of dihydrostreptomycin and neomycin for 323 Salmonella, 178 Escherichia coli and twenty-six Pasteurella multocida isolates recovered from newborn calves. Apramycin exhibited better in vitro anti-bacterial activity than dihydrostreptomycin and neomycin; isolates of Salmonella group B and E. coli resistant to the latter were sensitive to apramycin. The two-compartment open model was appropriate for the analysis of serum apramycin concentrations measured after intravenous (i.v.) administration. The distribution half-life (t 1/2 alpha) of the drug was 28 min, the elimination half-life (t 1/2 beta) was 4.4 h, and the apparent volume of distribution (V1) and the distribution volume at steady state (Vdss) were 0.34 and 0.71 l/kg, respectively. The drug was quickly and completely absorbed after intramuscular (i.m.) injection; peak serum drug concentrations were directly related to the dose administered, they were obtained 1-2 h after treatment and the i.m. t 1/2 beta was 5 h. There was no evidence of drug accumulation in the serum after three daily i.m. injections at 20 mg/kg. More than 95% of the i.v. and i.m. doses were recovered in the urine within 96 h post-treatment but the cumulative percentage of drug recovery in the urine after oral treatment was 11%. The durations of free drug concentrations in the tissues after i.v. and i.m. injection were estimated from the serum drug level data, percent of serum protein binding, Vdss, t 1/2 beta, and the MIC. Computations showed that apramycin should be administered i.m. at 20 mg/kg every 24 h in order to maintain in tissues potentially effective drug concentrations sufficient to inhibit 50% of the Salmonella, E. coli, and P. multocida isolates, and at 12-h intervals to inhibit 90% of the isolates.


Subject(s)
Anti-Bacterial Agents/metabolism , Cattle/metabolism , Nebramycin/metabolism , Animals , Bacterial Infections/veterinary , Cattle Diseases/prevention & control , Female , Injections, Intramuscular , Injections, Intravenous , Kinetics , Male , Nebramycin/administration & dosage , Nebramycin/analogs & derivatives , Nebramycin/blood
14.
Antimicrob Agents Chemother ; 14(1): 69-72, 1978 Jul.
Article in English | MEDLINE | ID: mdl-356726

ABSTRACT

Examination of a number of R-plasmid-containing bacterial isolates of animal origin has revealed the presence of a new aminoglycoside acetyltransferase (3-N) with a broad substrate range that includes all the disubstituted 2-deoxystreptamine antibiotics and also the novel monosubstituted antibiotic apramycin. Antibiotic derivatives acylated with hydroxyaminobutyric acid at the 1-amino position were not modified by the enzyme.


Subject(s)
Acetyltransferases/physiology , Anti-Bacterial Agents/pharmacology , Escherichia coli/enzymology , Nebramycin/pharmacology , Drug Resistance, Microbial , Escherichia coli/drug effects , Nebramycin/analogs & derivatives , Nebramycin/metabolism
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