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D L Oxender

Publications and source records attributed to D L Oxender.

At least 37 records · Page 2Linked to original sources
PubMed1991

A bacterial system for investigating transport effects of cystic fibrosis--associated mutations.

LIV-I, a high-affinity system that transports neutral, branched-chain amino acids into Escherichia coli, has two components, LivG and LivF, that are homologous to the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). CF-associated mutations of human CFTR were introduced into corresponding regions of LivG, and their effects on leucine transport could be grouped into three classes. Mutations were found that (i) abolished LIV-I--directed transport, (ii) retained about a quarter of wild-type activity at the Michaelis-Menten constant (KM), and (iii) had minimal activity at the KM. A mutation equivalent to a benign polymorphism had no effect on transport. The correlation of these mutational phenotypes in LivG and CFTR suggests that the LIV-I prokaryotic transporter is functionally similar to the CF protein and that this similarity can be exploited to clarify the properties of the nucleotide-binding fold in this superfamily of proteins.

ATP-Binding Cassette Transporters↗
PubMed1991

Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli.

Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.

Bacterial Proteins↗
PubMed1991

Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli.

The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli. The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C-terminal deletions or fusions of the livK gene to trpA (encoding the alpha subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the alpha-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.

Amino Acid Sequence↗
PubMed1991

Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.

Alkaline Phosphatase↗
PubMed1990

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli.

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.

Amino Acid Sequence↗
PubMed1990

A novel phosphate-regulated expression vector in Escherichia coli.

The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.

Gene Expression Regulation, Bacterial↗
PubMed1990

Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli.

A role for the leucyl-tRNA synthetase (EC 6.1.1.4) has been established for regulating the transport of leucine across the inner membrane of Escherichia coli by the leucine, isoleucine, valine (LIV-I) transport system. This transport system is mediated by interactions of periplasmic binding proteins with a complex of membrane-associated proteins, and transcription of the high-affinity branched-chain amino acid transport system genes is repressed by growth of E. coli on high levels of leucine. We now report results from sequence comparisons and structural modeling studies, which indicate that the leucine-specific binding protein, one of the periplasmic components of the LIV-I transport system, contains a 121-residue stretch, representing 36% of the mature protein, which displays both sequence and structural similarities to a region within the putative nucleotide-binding domain of leucyl-tRNA synthetase. Early fusion events between ancestral genes for the leucine-specific binding protein and leucyl-tRNA synthetase could account for the similarity and suggest that processes of aminoacylation and transport for leucine in E. coli may be performed by evolutionarily interrelated proteins.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L.

The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.

Amino Acid Transport Systems↗
PubMed1988

Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli.

Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that form the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of threonine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two- and four-fold, respectively. Replacement of arginine 54 by histidine (RH84) or glycine 85 by arginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants produces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimers upon treatment at 65 degrees C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Second-site revertants of Escherichia coli trp repressor mutants.

Second-site reversion studies were performed with five missense mutants with defects in the trp repressor of Escherichia coli. These mutants were altered throughout the gene. The same unidirectional mutagen used in the isolation of these mutants, hydroxylamine, was used in reversion studies, to increase the likelihood that the revertants obtained would have second-site changes. Most of the second-site revertants were found to have the same amino acid substitutions detected previously as superrepressor changes. These second-site revertant repressors were more active in vivo than their parental mutant repressors, in the presence or absence of exogenous tryptophan. Apparently superrepressor changes at many locations in this protein can act globally to increase the activity of mutant repressors.

Bacterial Proteins↗
PubMed1986

Cloning, expression, and nucleotide sequence of livR, the repressor for high-affinity branched-chain amino acid transport in Escherichia coli.

The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106. The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations. Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment. Expression of the pANT plasmids in E. coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000. DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155. The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices. These results suggest that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.

Amino Acid Sequence↗
PubMed1986

Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli.

The physical location of the genetically defined livH gene was mapped in the 17-kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis. These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein. Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined. The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene. The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure. The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues. Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product.

Amino Acid Sequence↗