Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Colicin M (Cma) displays a unique activity that interferes with murein and O-antigen biosynthesis through inhibition of lipid-carrier regeneration. Immunity is conferred by a specific immunity protein (Cmi) that inhibits the action of colicin M in the periplasm. The subcellular location of Cmi was determined by constructing hybrid proteins between Cmi and the TEM-beta-lactamase (BlaM), which confers resistance to ampicillin only when it is translocated across the cytoplasmic membrane with the aid of Cmi. The smallest Cmi'-BlaM hybrid that conferred resistance to 50 micrograms/ml ampicillin contained 19 amino acid residues of Cmi; cells expressing Cmi'-BlaM with only five N-terminal Cmi residues were ampicillin sensitive. These results support a model in which the hydrophobic sequence of Cmi comprising residues 3-23 serves to translocate residues 24-117 of Cmi into the periplasm and anchors Cmi to the cytoplasmic membrane. Residues 8-23 are integrated in the cytoplasmic membrane and are not involved in Cma recognition. This model was further tested by replacing residues 1-23 of Cmi by the hydrophobic amino acid sequence 1-42 of the penicillin binding protein 3 (PBP3). In vivo, PBP3'-'Cmi was as active as Cmi, demonstrating that translocation and anchoring of Cmi is not sequence-specific. Substitution of the 23 N-terminal residues of Cmi by the cleavable signal peptide of BlaM resulted in an active BlaM'-'Cmi hybrid protein. The immunity conferred by BlaM'-'Cmi was high, but not as high as that associated with Cmi and PBP3'-'Cmi, demonstrating that soluble Cmi lacking its membrane anchor is still active, but immobilization in the cytoplasmic membrane, the target site of Cma, increases its efficiency. Cmi delta 1-23 remained in the cytoplasm and conferred no immunity. We propose that the immunity protein inactivates colicin M in the periplasm before Cma can reach its target in the cytoplasmic membrane.
Mol Gen Genet 1996 Jun 12
PMID:Colicin M is inactivated during import by its immunity protein. 867 83

The anion exchange system of human red blood cells can be completely inhibited by a large number of arginine-specific reagents. Kinetic studies have shown that these reagents act on the substrate binding site. Complete inhibition of the transport system with 14C phenylglyoxal is accompanied by modification of 2 to 3 arginine residues on the transmembrane segment of band 3. The inhibition and the binding of 14C phenylglyoxal to band 3 is reduced significantly in presence of chloride ions. The interactions between the reversible competitive inhibitor, 4-hydroxy-3-nitrophenylglyoxal (HNPG) and other irreversible anion transport inhibitors with well localized binding sites have been studied. A positive cooperativity between HNPG binding and the inhibition caused by eosin-5-maleimide (EMA), has been measured. Nearly no interactions have been found between HNPG binding site and the histidine residue(s) which react with diethylpyrocarbonate (DEPC). Sulphate self-exchange is irreversibly inhibited by two carboxyl-group reagents, 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimde (EDC) and N'-(3-dimethylaminopropyl)-N-ethylcarbodiimid methoiodide (EAC), studies on their interaction with the HNPG binding site gave results showing that the reaction site(s) of these reagents do not overlap and are not adjacent to the essential arginine(s). A model concerning such interactions is discussed.
Cell Mol Biol (Noisy-le-grand) 1996 Nov
PMID:Chemical labelling of arginyl-residues involved in anion transport mediated by human band 3 protein and some aspects of its location in the peptide chain. 896 Jul 80

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and beta-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as "TEM-1B like" and "TEM-2 like" restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the beta-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of beta-lactam-beta-lactamase inhibitor combinations.
J Mol Evol 1997 Jan
PMID:Molecular diversity and evolution of blaTEM genes encoding beta-lactamases resistant to clavulanic acid in clinical E. coli. 901 Jan 36

The presence of a tumour significantly changes nitrogen metabolism, including that of amino acids and polyamines, in host animals. In this study, we examine whether developing tumours affect the metabolic relationship of arginine and ornithine, precursors of polyamines, in the testosterone-induced hypertrophic mouse kidney model. Androgen-induced changes in the activity of enzymes involved with ornithine biosynthesis (arginase), its consumption (ornithine aminotransferase, OAT and ornithine decarboxylase, ODC) and the hypertrophy of host mouse kidney were not affected by the presence of an ascitic tumour (EAC) and only slightly by a mammary carcinoma (MaCa). The HPLC determined renal level of arginine and ornithine showed a striking homeostasis and was disturbed neither by testosterone nor EAC. The effect of MaCa and testosterone on the levels of both amino acids, although significant, was not very pronounced. Developing tumours, especially ascitic, altered the renal activity of OAT and ODC, but not of arginase, in testosterone-untreated mice. All examined tumours, EAC, L 1210 and MaCa actively metabolized arginine and ornithine. the tumour content of arginine which coincided with the activity of arginase, resulted in a marked increase of the ornithine/arginine ratio in tumours, when compared with kidneys. These results indicate that the androgen-induced anabolic response in mouse kidney is preserved, in spite of tumour requirements for essential metabolites.
Mol Cell Biochem 1997 Mar
PMID:Tumour effect on arginine/ornithine metabolic relationship in hypertrophic mouse kidney. 906 93

Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA are packaged in multi-component large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200 S region in sucrose gradients, contain all U small nuclear RNPs required for precursor messenger RNA (pre-mRNA) splicing and several protein splicing factors, including U2AF and the SR proteins. Electron microscopy of lnRNP particles revealed a large compact structure of 50 nm in diameter. In this study we employed automated computed tomography from electron micrographs for the three-dimensional (3D) image reconstruction of individual lnRNP particles isolated from mammalian cells nuclei and negatively stained. For each particle, a tilt series of 71 images was collected by direct digital recording of the images on a CCD camera attached to a computer controlled TEM facility. The 3D image was reconstructed according to the back projection principle. For rendering, real time display and comparison of the reconstructed particles, interactive computer graphics was employed. The reconstructed 3D images show a compact structure composed of four major subunits connected to each other. Comparison of the reconstructed lnRNP particles revealed morphological similarity of the individual particles, as well as similarity among the sub-structures. Based on these observations we propose a model for the packaging of nuclear pre-mRNAs in lnRNP particles where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multi-intronic pre-mRNAs, and with the fact that the splicing of multi-intronic pre-mRNAs does not occur in a sequential manner.
J Mol Biol 1997 Apr 04
PMID:Three-dimensional image reconstruction of large nuclear RNP (lnRNP) particles by automated electron tomography. 912 39

The crystal structure of a phosphonate complex of the class A TEM-1 beta-lactamase has been determined to a resolution of 2.0 A. The phosphonate appears stoichiometrically at the active site, bound covalently to Ser70Ogamma, with one phosphonyl oxygen in the oxyanion hole. Although the overall structure is very similar to that of the native enzyme (rms difference 0.37 A for all heavy atoms), changes have occurred in the position of active site functional groups. The active site is also not in the conformation observed in the complex of another class A beta-lactamase, that of Staphylococcus aureus PC1, with the same phosphonate [Chen, C. C. H., et al. (1993) J. Mol. Biol. 234,165-178]. Both phosphonate structures, however, can be seen to represent models of acylation transition-states since in each the deacylating water molecule appears firmly bound to the Glu166 carboxylate group. The major difference between the structures lies in the positioning of Lys73Nzeta and Ser130Ogamma. In the S. aureus structure, the closest interaction of these functional groups is between Lys73Nzeta and Ser70Ogamma (2.8 A), while in the TEM-1 structure it is between Ser130Ogamma and the second phosphonyl oxygen of the bound inhibitor (2.8 A). The former structure therefore may resemble a transition state for formation of the tetrahedral species in acylation by nucleophilic attack on the substrate, where Lys73Nzeta presumably catalyzes the reaction as a general base. The TEM-1 structure can then be seen as an analogue of the transition state for breakdown of the tetrahedral species, where Ser130Ogamma is acting as a general acid, assisting the departure of the leaving group. The class A beta-lactamase crystal structures now available lead to a self-consistent proposal for a mechanism of catalysis by these enzymes.
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PMID:Crystal structure of an acylation transition-state analog of the TEM-1 beta-lactamase. Mechanistic implications for class A beta-lactamases. 948 12

This review traces some of the key features of the folding of beta-lactamases and their relevance to the way proteins fold in general. Studies on the enzymes have highlighted the nature and role of equilibrium and transient condensed states. The kinetics of folding are multiphasic, and when monitored by acrylamide quenching of the tryptophan fluorescence, an early phase provides evidence for the transient accumulation of a nonnative intermediate involving burial of tryptophan in a nonpolar environment. Intermediate phases can be understood in terms of progressive folding of different parts of the molecule. The later, slow phases are associated with proline isomerization in the TEM-1 enzyme and, in its P167T mutant form, with isomerization from trans to cis of the E166 T167 peptide bond. Coupled with kinetic and X-ray crystallographic studies of the beta-lactamase from Staphylococcus aureus and its D179Q mutant, it appears that the final stage of folding is that of collapse and packing of the omega-loop on to the main body of the protein.
Cell Mol Life Sci 1998 Apr
PMID:Beta-lactamases as models for protein-folding studies. 961 75

In the present study we analyse the nature and the functional significance of the spherical and fibrillo-granular structures appearing in the oocyte nucleus of the lizard Podarcis sicula, following the disappearance of the typical nucleolus. By LM and TEM approaches, we demonstrate that the fibrillo-granuli, containing DNA, RNA and nucleolar proteins, are micronucleoli transcriptionally active and that their DNA is probably derived from nucleolar fragmentation. By contrast, we could not explain the origin and role of the so-called spherical bodies, appearing earlier in oocyte growth; these, in fact, do not contain nucleic acids or nucleolar proteins and do not incorporate uridine. Different possible explanations of their significance are discussed.
Mol Reprod Dev 1998 Dec
PMID:Structural and functional modifications of the nucleolus during previtellogenic oocyte growth in the lizard Podarcis sicula. 982 Feb

For a number of years a major limitation in genetic analysis of protein function has been the inability to introduce multiple substitutions at distant sites that would enable the selection of clusters of mutations required for improved or novel biological functions. In order to achieve this, we have recently developed a novel mutagenesis procedure in which the triphosphate derivatives of a pyrimidine (6-(2-deoxy-beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one; dP) and a purine (8-oxo-2'-deoxyguanosine; 8-oxodG) nucleoside analogue are employed in DNA synthesis reactions in vitro. The procedure allows control of the mutational load and can yield frequencies of amino acid residue substitutions at least one order of magnitude greater than those previously achieved. Here we report the results of an experiment in which we have hypermutated the bacterial enzyme TEM-1 beta-lactamase and selected small pools (<1.5x10(5)) of clones for enzymatic activity against the beta-lactam antibiotic cefotaxime. The experiment resulted in the isolation of a number of TEM-1 mutants with greatly improved activity against cefotaxime. Among these, clone 3D.5 (E104K:M182T:G238S) exhibited a minimum inhibitory concentration for cefotaxime 20,000-fold higher than wild-type TEM-1 and a catalytic efficiency (kcat/Km) 2383 times higher than the wild-type enzyme. Thus, small pools of hypermutated sequences enabled the selection of one of the most active extended beta-lactamases described so far. These results argue against the accepted view that multiple rounds of low-rate mutagenesis and stepwise selection are essential for in vitro protein evolution and extend the scope of directed molecular evolution to proteins for which no genetic selection is available.
J Mol Biol 1999 Jan 15
PMID:The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1 beta-lactamase. 987 43

A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue.
J Mol Biol 2000 Jan 21
PMID:Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop. 1062 44


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