Purification, characterization, and investigation of the mechanism of aminoglycoside 3'-phosphotransferase type Ia.


:Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coli harboring APH-(3')-Ia were determined. The enzyme phosphorylates its substrates with kcat/Km values of 10(6)-10(8) M-1 s-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0-7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phosphorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water ("ATP hydrolase" activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on kcat/Km) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium-random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia.






Siregar JJ,Miroshnikov K,Mobashery S




Has Abstract


1995-10-03 00:00:00












  • Structural evidence: a single charged residue affects substrate binding in cytochrome P450 BM-3.

    abstract::Cytochrome P450 BM-3 is a bacterial enzyme with sequence similarity to mammalian P450s that catalyzes the hydroxylation of fatty acids with high efficiency. Enzyme-substrate binding and dynamics has been an important topic of study for cytochromes P450 because most of the crystal structures of substrate-bound structur...


    pub_type: 杂志文章


    authors: Catalano J,Sadre-Bazzaz K,Amodeo GA,Tong L,McDermott A

    更新日期:2013-10-01 00:00:00

  • Effects of chromogranin expression on inositol 1,4,5-trisphosphate-induced intracellular Ca2+ mobilization.

    abstract::We show here that expression of chromogranins in non-neuroendocrine NIH3T3 cells significantly increased the amount of IP(3)-mediated intracellular Ca(2+) mobilization in these cells, whereas suppression of them in neuroendocrine PC12 cells decreased the amount of mobilized Ca(2+). We have therefore investigated the r...


    pub_type: 杂志文章


    authors: Huh YH,Jeon SH,Yoo JA,Park SY,Yoo SH

    更新日期:2005-04-26 00:00:00

  • Molecular mobility of the Ca(2+)-deficient EF-hand of cardiac troponin C as revealed by fluorescence polarization of genetically inserted tryptophan.

    abstract::To probe attitudinal features of the Ca(2+)-deficient site (site I) in the Ca2+ switch of cardiac troponin C (cTnC), we have examined steady-state fluorescence emission and polarization of a Trp26 inserted in a recombinant cardiac TnC (cTnC3.W) and compared these with the properties of the Ca(2+)-competent site I in s...


    pub_type: 杂志文章


    authors: Rao VG,Akella AB,Su H,Gulati J

    更新日期:1995-01-17 00:00:00

  • Kinetic evidence for folding and unfolding intermediates in staphylococcal nuclease.

    abstract::The complex kinetic behavior commonly observed in protein folding studies suggests that a heterogeneous population of molecules exists in solution and that a number of discrete steps are involved in the conversion of unfolded molecules to the fully native form. A central issue in protein folding is whether any of thes...


    pub_type: 杂志文章


    authors: Walkenhorst WF,Green SM,Roder H

    更新日期:1997-05-13 00:00:00

  • Human erythrocytic purine nucleoside phosphorylase: reaction with sugar-modified nucleoside substrates.

    abstract::The kinetic parameters (Km and Vmax) of sugar-modified analogues of inosine and guanosine have been determined with human erythrocytic purine nucleoside phosphorylase (PNP). Steric alterations at the 2' and 3' positions greatly lessened or abolished substrate activity. However, the 5'-deoxy- and 2',5'-dideoxy-beta-D-r...


    pub_type: 杂志文章


    authors: Stoeckler JD,Cambor C,Parks RE Jr

    更新日期:1980-01-08 00:00:00

  • Catalytic properties of hairpin ribozymes derived from Chicory yellow mottle virus and arabis mosaic virus satellite RNAs.

    abstract::Regions of the negative strands of the satellite RNAs of chicory yellow mottle virus (sCYMV1) and arabis mosaic virus (sArMV) have similarity in sequence and predicted secondary structure compared to the tobacco ringspot virus satellite RNA (sTRSV) hairpin ribozyme, suggesting that they may also be catalytic RNAs of a...


    pub_type: 杂志文章


    authors: DeYoung M,Siwkowski AM,Lian Y,Hampel A

    更新日期:1995-12-05 00:00:00

  • Neutral glycosphingolipids in hairy cell leukemia.

    abstract::The neutral glycosphingolipids of hairy cells from a patient with hairy cell leukemia were chemically analyzed by thin-layer and gas-liquid chromatography, mass spectrometry, combined gas chromatography-mass spectrometry, and glycosidase treatment. These cells were found to have compounds containing one to four sugars...


    pub_type: 杂志文章


    authors: Lee WM,Klock JC,Macher BA

    更新日期:1981-10-27 00:00:00

  • Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH.

    abstract::Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same con...


    pub_type: 杂志文章


    authors: Haas DD,Ware BR

    更新日期:1978-11-14 00:00:00

  • Apical membrane of the gastric parietal cell: water, proton, and nonelectrolyte permeabilities.

    abstract::Gastric parietal cell apical membranes must protect the cell from the extremely low pH and wide variations in osmolality of the gastric juice. To characterize the permeability properties of gastric apical membranes, we have measured passive permeabilities to water, protons, NH3, and small nonelectrolytes of membrane v...


    pub_type: 杂志文章


    authors: Priver NA,Rabon EC,Zeidel ML

    更新日期:1993-03-16 00:00:00

  • Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities.

    abstract::Ultraviolet irradiation of DNA results in various pyrimidine modifications. We studied the excision of an ultraviolet thymine photoproduct by Escherichia coli endonuclease III and by a preparation of human WI-38 cells. These enzymes cleave UV-irradiated DNA at apyrimidinic sites formed by glycosylic removal of the pho...


    pub_type: 杂志文章


    authors: Ganguly T,Weems KM,Duker NJ

    更新日期:1990-08-07 00:00:00

  • Cooperative binding of midazolam with testosterone and alpha-naphthoflavone within the CYP3A4 active site: a NMR T1 paramagnetic relaxation study.

    abstract::Recent studies have indicated that CYP3A4 exhibits non-Michaelis-Menten kinetics for numerous substrates. Both homo- and heterotropic activation have been reported, and kinetic models have suggested multiple substrates within the active site. We provide some of the first physicochemical data supporting the hypothesis ...


    pub_type: 杂志文章


    authors: Cameron MD,Wen B,Allen KE,Roberts AG,Schuman JT,Campbell AP,Kunze KL,Nelson SD

    更新日期:2005-11-01 00:00:00

  • Molecular modeling of the multidomain structures of the proteoglycan binding region and the link protein of cartilage by neutron and synchrotron X-ray scattering.

    abstract::The interaction of proteoglycan monomers with hyaluronate in cartilage is mediated by a globular binding region at the N-terminus of the proteoglycan monomer; this interaction is stabilized by link protein. Sequences show that both the binding region (27% carbohydrate) and the link protein (6% carbohydrate) contain an...


    pub_type: 杂志文章


    authors: Perkins SJ,Nealis AS,Dunham DG,Hardingham TE,Muir IH

    更新日期:1991-11-05 00:00:00

  • Photooxidation pathway of chlorophyll Z in photosystem II as Studied by Fourier transform infrared spectroscopy.

    abstract::The oxidation pathway of chlorophyll Z (ChlZ) in photosystem II (PSII) at cryogenic temperatures was studied by means of light-induced Fourier transform infrared (FTIR) difference spectroscopy. To examine the involvement of redox-active beta-carotene (Car) in the pathway, two Car molecules in Mn-depleted PSII membrane...


    pub_type: 杂志文章


    authors: Kitajima Y,Noguchi T

    更新日期:2006-02-14 00:00:00

  • Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage.

    abstract::Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a...


    pub_type: 杂志文章


    authors: Codreanu SG,Adams DG,Dawson ES,Wadzinski BE,Liebler DC

    更新日期:2006-08-22 00:00:00

  • Interaction of the fluorescence-labeled secretory component with human polymeric immunoglobulins.

    abstract::The secretory component (SC) isolated from human milk was labeled with 2 mol of the fluorescent thiol reagent N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide (DACM) per mol of SC through the reactive disulfide bond of SC. The binding of the labeled SC to polymeric immunoglobulins was examined by gel filtration by me...


    pub_type: 杂志文章


    authors: Goto Y,Aki K

    更新日期:1984-12-18 00:00:00

  • Physical and conformational properties of synthetic idealized signal sequences parallel their biological function.

    abstract::Transported proteins often contain an extension sequence called the signal peptide. The alkaline phosphatase (PhoA) signal sequence represents a typical signal peptide for comparison to idealized sequences both in vivo and in vitro. We have designed a series of idealized signal sequences which vary in amino terminal c...


    pub_type: 杂志文章


    authors: Izard JW,Doughty MB,Kendall DA

    更新日期:1995-08-08 00:00:00

  • Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.

    abstract::[15N]Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in order to produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resona...


    pub_type: 杂志文章


    authors: Campbell-Burk S

    更新日期:1989-11-28 00:00:00

  • Ca(2+)-dependent binding of endonexin (annexin IV) to membranes: analysis of the effects of membrane lipid composition and development of a predictive model for the binding interaction.

    abstract::Endonexin (annexin IV) is a member of the annexin family of homologous proteins that bind membranes and aggregate vesicles in a calcium-dependent fashion. This study examines the lipid modulation and mechanism of the binding of endonexin to membranes using a fluorescence energy transfer assay to measure bovine endonex...


    pub_type: 杂志文章


    authors: Junker M,Creutz CE

    更新日期:1994-08-02 00:00:00

  • The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis.

    abstract::trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocr...


    pub_type: 杂志文章


    authors: Azurmendi HF,Wang SC,Massiah MA,Poelarends GJ,Whitman CP,Mildvan AS

    更新日期:2004-04-13 00:00:00

  • High-density-lipoprotein-induced cholesterol efflux from arterial smooth muscle cell derived foam cells: functional relationship of the cholesteryl ester cycle and eicosanoid biosynthesis.

    abstract::Eicosanoids have been implicated in the regulation of arterial smooth muscle cell (SMC) cholesteryl ester (CE) metabolism. These eicosanoids, which include prostacyclin (PGI2), stimulate CE hydrolytic activities. High-density lipoproteins (HDL), which promote cholesterol efflux, also stimulate PGI2 production, suggest...


    pub_type: 杂志文章


    authors: Pomerantz KB,Hajjar DP

    更新日期:1990-02-20 00:00:00

  • Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus.

    abstract::Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochlor...


    pub_type: 杂志文章


    authors: Xu X,Liu Q,Xie Y

    更新日期:2002-03-19 00:00:00

  • Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides.

    abstract::Effects of different end sequences on melting, circular dichroism spectra (CD), and enzyme binding properties were investigated for four 40 base pair, non-self-complementary duplex DNA oligomers. The center sequences of these oligoduplexes have either of two 22 base pair modules flanked on both sides by sequences diff...


    pub_type: 杂志文章


    authors: Vallone PM,Benight AS

    更新日期:2000-07-04 00:00:00

  • Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor.

    abstract::The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entir...


    pub_type: 杂志文章


    authors: Pan T,Freedman LP,Coleman JE

    更新日期:1990-10-02 00:00:00

  • Stabilizing and Understanding a Miniprotein by Rational Redesign.

    abstract::Miniproteins reduce the complexity of the protein-folding problem allowing systematic studies of contributions to protein folding and stabilization. Here, we describe the rational redesign of a miniprotein, PPα, comprising a polyproline II helix, a loop, and an α helix. The redesign provides a de novo framework for in...


    pub_type: 杂志文章


    authors: Porter Goff KL,Nicol D,Williams C,Crump MP,Zieleniewski F,Samphire JL,Baker EG,Woolfson DN

    更新日期:2019-07-16 00:00:00

  • Structural basis of DNA recognition and mechanism of quadruplex formation by the beta subunit of the Oxytricha telomere binding protein.

    abstract::Interactions of the beta subunit of the Oxytricha nova telomere binding protein with the telomeric DNA sequences, d(T4G4)2 and dT6(T4G4)2, have been investigated in vitro using Raman and fluorescence spectroscopies. Raman difference spectra show that the beta subunit binds to both d(T4G4)2 and dT6(T4G4)2 but promotes ...


    pub_type: 杂志文章


    authors: Laporte L,Thomas GJ Jr

    更新日期:1998-02-03 00:00:00

  • The alpha-helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer of fatty acids to phospholipid membranes.

    abstract::Intestinal fatty acid binding protein (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms for the transfer of fatty acids (FAs) to acceptor membranes. Transfer from IFABP occurs during protein-membrane collisional interaction...


    pub_type: 杂志文章


    authors: Córsico B,Liou HL,Storch J

    更新日期:2004-03-30 00:00:00

  • One-step purification of Actinoplanes missouriensis D-xylose isomerase by high-performance immobilized copper-affinity chromatography: functional analysis of surface histidine residues by site-directed mutagenesis.

    abstract::D-Xylose isomerase (XI) is a heat-stable homotetrameric enzyme used in industry for the production of high-fructose corn syrups by isomerization of D-glucose into D-fructose. To carry out biochemical and structural studies of this enzyme and of its engineered variants, a rapid and convenient method of purification of ...


    pub_type: 杂志文章


    authors: Mrabet NT

    更新日期:1992-03-17 00:00:00

  • Comparative EPR and redox studies of three prokaryotic enzymes of the xanthine oxidase family: quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase.

    abstract::For three prokaryotic enzymes of the xanthine oxidase family, namely quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase, the electron transfer centers were investigated by electron paramagnetic resonance. The enzymes are containing a molybdenum-molybdopterin cytosine dinucleotide cofac...


    pub_type: 杂志文章


    authors: Canne C,Stephan I,Finsterbusch J,Lingens F,Kappl R,Fetzner S,Hüttermann J

    更新日期:1997-08-12 00:00:00

  • Structural insights into the membrane fusion mechanism mediated by influenza virus hemagglutinin.

    abstract::Membrane fusion is involved in many fundamental cellular processes and entry of enveloped viruses into host cells. Influenza type A virus HA has long served as a paradigm for mechanistic studies of protein-mediated membrane fusion via large-scale structural rearrangements induced by acidic pH. Here we report the newly...


    pub_type: 杂志文章


    authors: Ni F,Chen X,Shen J,Wang Q

    更新日期:2014-02-11 00:00:00

  • Ligand binding alters the backbone mobility of intestinal fatty acid-binding protein as monitored by 15N NMR relaxation and 1H exchange.

    abstract::The backbone dynamics of the liganded (holo) and unliganded (apo) forms of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) have been characterized and compared using amide 15N relaxation and 1H exchange NMR measurements. The amide 1H/15N resonances for apo and holo I-FABP were assigned at 2...


    pub_type: 杂志文章


    authors: Hodsdon ME,Cistola DP

    更新日期:1997-02-25 00:00:00