Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.
ABSTRACT The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.
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ABSTRACT: Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.Biochimica et Biophysica Acta 01/1979; 521(2):459-69. · 4.66 Impact Factor
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ABSTRACT: In Escherichia coli cell-free systems, the aminboglycoside antibiotic hygromycin B inhibits (80%) the elogation-factor-(EF)G-plus-GTP-dependent reaction of either AcPhe-tRNA or natural peptideyl-tRNA with puromycin. The antibiotic does not substantially affect the poly(uridylic acid)-directed, EF-Tu-plus-GTP-dependent binding of Phe-tRNA to ribosomes or the reaction of donor-site-bound AcPhe-tRNA with puromycine. Moreover, the nascnet peptide chains of either purified endogenous E. coli polysomes or poly(uridylic acid)-programmed ribosomes, blocked by hygromycin B during polypeptide synthesis, react slowly with puromycin while the chains of uninhibited polysomes or tetracycline-blocked ribosomes react very rapidly. These results indicate that hygromyckin B temporaily retains peptidely-tRNA in the ribosomal acceptor site and, consequently, that the inhibition of polypeptide chain elongation by the antibiotic is, at least in part, due to the impairment of ribosomal translocation.Hygromycin B at concentrations strongly inhibitory for translocation (80 μM) does not affect the EF-G-plus-ribosome-dependent GTP hydrolysis, the formation of the GDP · EF-G · ribosome · fusidic-acid complex or the non-enzyme binding of AcPhe-tRNA to the ribosomal donor site. In contrast, hygromycin B inhibits the non-enzymic translocation and the release of AcPhe-tRNA from the ribosomal acceptor site promoted by depletion of NH+4 ions. It is suggested that hygromycin B inhibits the movement of peptideyl-tRNA associated with translocation by interfering with its exit from the ribosomal acceptor site.Hygromycin B has been previously described as an inducer of misreading [Davies, J., Gorini, L. and Davis, B. D. (1965) Mol. Pharmacol. 1, 93–106]. It is now shown that the antibiotic interfaces with translation and induces misreading in the same poly(uridylic acid)-directed system and in approximately the same range of concentrations. The results suggest a relationship between the ribosomal processes of aminoacyl-tRNA recognition and translocation.European Journal of Biochemistry. 05/1978; 87(1):21 - 27.
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ABSTRACT: Enterococcus casseliflavus UC73 is a clinical blood isolate with high-level resistance to gentamicin. DNA preparations from UC73 failed to hybridize with intragenic probes for aac(6')-Ie-aph(2'')-Ia and aph(2'')-Ic. A 4-kb fragment from UC73 was cloned and found to confer resistance to gentamicin in Escherichia coli DH5alpha transformants. Nucleotide sequence analysis revealed the presence of a 906-bp open reading frame whose deduced amino acid sequence had a region with homology to the aminoglycoside-modifying enzyme APH(2'')-Ic and to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2''). The gene is designated aph(2'')-Id, and its observed phosphotransferase activity is designated APH(2'')-Id. A PCR-generated intragenic probe hybridized to the genomic DNA from 17 of 118 enterococcal clinical isolates (108 with high-level gentamicin resistance) from five hospitals. All 17 were vancomycin-resistant Enterococcus faecium isolates, and pulsed-field typing revealed three distinct clones. The combination of ampicillin plus either amikacin or neomycin exhibited synergistic killing against E. casseliflavus UC73. Screening and interpretation of high-level aminoglycoside resistance in enterococci may need to be modified to include detection of APH(2'')-Id.Antimicrobial Agents and Chemotherapy 06/1998; 42(5):1229-32. · 4.57 Impact Factor