scholarly journals Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport

1989 ◽  
Vol 258 (2) ◽  
pp. 389-396 ◽  
Author(s):  
I Yamato ◽  
Y Anraku
Keyword(s):  
Escherichia Coli ◽  
Substrate Binding ◽  
Ph Dependence ◽  
Plasmid Vector ◽  
Binary Complex ◽  
Wild Type ◽  
Transport Reaction ◽  
Substrate Transport ◽  
Vector Pbr322 ◽  
Dissociation Step

The lacYun gene, which encodes a lactose carrier showing the uncoupled phenotype of substrate transport in Escherichia coli [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414], was cloned on a plasmid vector, pBR322. The binding of a substrate, p-nitrophenyl alpha-galactoside, to the lacYun carrier in membranes from the strain harbouring the lacYun clone showed a pH-dependence different from its binding to the wild-type lactose carrier. This finding indicated that the lacYun mutation confers higher affinity for H+ on the carrier, exerting its effect on the less efficient dissociation of substrate inside cells. The result coincides with the proposal [Yamato & Rosenbusch (1983) FEBS Lett. 151, 102-104] that the proton affecting the substrate binding is the coupling proton of the proton/lactose symport reaction, which allows only the ordered mechanism of binding of substrate to an H+-carrier binary complex. From the simplest model of the symport reaction, constructed on the basis of these results, the coupling site of energy in the carrier cycle of the transport reaction can be identified at the substrate-dissociation step inside cells.

scholarly journals In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1451-1459 ◽  
Author(s):  
Bernadette L. LaMonte ◽  
Jeffrey A. Hughes
Keyword(s):  
Escherichia Coli ◽  
Plasmid Vector ◽  
Wild Type ◽  
Methionine Biosynthesis ◽  
Induced Expression ◽  
Defined Media ◽  
Sam Synthetase ◽  
Hydrolysis Of

Regulation of methionine biosynthesis in Escherichia coli involves a complex of the MetJ aporepressor protein and S-adenosylmethionine (SAM) repressing expression of most genes in the met regulon. To test the role of SAM in the regulation of met genes directly, SAM pools were depleted by the in vivo expression of the cloned plasmid vector-based coliphage T3 SAM hydrolase (SAMase) gene. Cultures with in vivo SAMase activity were assayed for expression of the metA, B, C, E, F, H, J, K and R genes in cells grown in methionine-rich complete media as well as in defined media with and without l-methionine. In vivo SAMase activity dramatically induced expression between 11- and nearly 1000-fold depending on the gene assayed for all but metJ and metH, and these genes were induced over twofold. metJ : : Tn5 (aporepressor defective) and metK : : Tn5 (SAM synthetase impaired; produces <5 % of wild-type SAM) strains containing in vivo SAMase activity produced even higher met gene activity than that seen in comparably prepared cells with wild-type genes for all but metJ in a MetJ-deficient background. The SAMase-mediated hyperinduction of metH in wild-type cells and of the met genes assayed in metJ : : Tn5 and metK : : Tn5 cells provokes questions about how other elements such as the MetR activator protein or factors beyond the met regulon itself might be involved in the regulation of genes responsible for methionine biosynthesis.

scholarly journals The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype

1985 ◽  
Vol 227 (1) ◽  
pp. 287-297 ◽  
Author(s):  
J K Wright ◽  
R Seckler
Keyword(s):  
Escherichia Coli ◽  
Active Transport ◽  
Rate Constants ◽  
Ph Dependence ◽  
Maximal Velocity ◽  
Chemiosmotic Theory ◽  
Wild Type ◽  
Terminal Sequence ◽  
Simple Interpretation

The Escherichia coli K12 strain X71-54 carries the lac YUN allele, coding for a lactose/H+ carrier defective in the accumulation of a number of galactosides [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414]. Previous studies proposed that the lower accumulation in the mutant be due to a faulty coupling of H+ and galactoside fluxes via the carrier. Immunochemical characterization of the carriers in membranes from mutant and parent strains with an antibody directed against the C-terminal decapeptide of the wild-type carrier leads to the conclusion that the mutant carrier is similar to the wild-type in terms of apparent Mr, C-terminal sequence, and level of incorporation into the membrane. The pH-dependence of galactoside transport was compared in the mutant and the parent. At pH 8.0-9.0, mutant and parent behave similarly with respect to the accumulation of beta-D-galactosyl 1-thio-beta-D-galactoside and to the ability to grow on the carrier substrate melibiose. At pH 6.0, both the maximal velocity for active transport and the level of accumulation of beta-D-galactosyl-1-thio-beta-D-galactoside are lower in the mutant. The mutant also is unable to grow on melibiose at pH 5.5. However, at pH 6.0 and low galactoside concentrations, the symport stoichiometry is 0.90 H+ per galactoside in the mutant as compared with 1.07 in the parent. These observations suggest that symport is normal in the mutant and that the lower rate of transport in the mutant is responsible for the phenotype. At higher galactoside concentrations, accumulation is determined not only thermodynamically but also kinetically, contrary to a simple interpretation of the chemiosmotic theory. Therefore lower rates of active transport can mimic the effect of uncoupling H+ and galactoside symport. Examination of countertransport in poisoned cells at pH 6.0 reveals that the rate constants for the reorientation of the loaded and unloaded carrier are altered in the mutant. The reorientation of the unloaded carrier is slower in the mutant. However, the reorientation of the galactoside-H+-carrier complex is slower for substrates like melibiose, but faster for substrates like lactose. These findings suggest that lactose-like and melibiose-like substrates interact with the carrier in slightly different ways.

Molecular cloning of the hamster papovavirus genome in Escherichia coli plasmid vector pBR322

Gene ◽  
1984 ◽  
Vol 29 (1-2) ◽  
pp. 243-246 ◽  
Author(s):  
Wolfgang Zimmermann ◽  
Hans Krause ◽  
Siegfried Scherneck ◽  
Jean Feunteun ◽  
Erhard Geissler
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Plasmid Vector ◽  
Coli Plasmid ◽  
Vector Pbr322

scholarly journals Role of Feedback Regulation of Pantothenate Kinase (CoaA) in Control of Coenzyme A Levels in Escherichia coli

2003 ◽  
Vol 185 (11) ◽  
pp. 3410-3415 ◽  
Author(s):  
Charles O. Rock ◽  
Hee-Won Park ◽  
Suzanne Jackowski
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Coenzyme A ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
Binary Complex ◽  
Wild Type ◽  
Pantothenate Kinase

ABSTRACT Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4′-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels.

scholarly journals Molecular cloning of pheR in Escherichia coli K-12

1982 ◽  
Vol 152 (1) ◽  
pp. 1-6
Author(s):  
J Gowrishankar ◽  
J Pittard
Keyword(s):  
Escherichia Coli ◽  
Tetracycline Resistance ◽  
Plasmid Vector ◽  
Chorismate Mutase ◽  
Gamma Delta ◽  
E Coli ◽  
Subunit Molecular Weight ◽  
K 12 ◽  
Vector Pbr322 ◽  
Pair Fragment

The regulator gene pheR, which in Escherichia coli controls the expression of pheA, the structural gene for chorismate mutase P-prephenate dehydratase, was cloned on to multicopy plasmids directly from the E. coli chromosome; this was achieved with the aid of the tetracycline resistance transposon, Tn10, that had been inserted very close to the pheR gene. Subsequently, pheR was subcloned on a 1.1-kilobase-pair fragment on the plasmid vector pBR322; its position on the plasmid was localized by the method of gamma delta-mediated transpositional inactivation. The pheR gene product was identified in maxicells and found to be a protein of subunit molecular weight 19,000, suggesting that the coding segment of the gene is about 500 nucleotide pairs long.

scholarly journals Revealing Escherichia coli type II l-asparaginase active site flexible loop in its open, ligand-free conformation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maristella Maggi ◽  
Massimiliano Meli ◽  
Giorgio Colombo ◽  
Claudia Scotti
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Substrate Binding ◽  
Water Molecules ◽  
Type Ii ◽  
Wild Type ◽  
Structural Element ◽  
Flexible Loop ◽  
Open Conformation ◽  
Ligand Free

AbstractSince 1993, when the structure of Escherichia coli type II l-asparaginase (EcAII) in complex with l-aspartate was firstly reported, many structures of the wild type and mutated enzyme have been deposited in the Protein Data Bank. None of them report the full structure of the monomer in its ligand-free, open conformation, mainly because of the high dynamic and flexibility of the active site flexible loop. Here we report for the first time the structure of EcAII wild type in its open conformation comprising, for at least one protomer, clear electron density for the active site flexible loop (PDB ID: 6YZI). The structural element is highly mobile and it is transposed onto the rigid part of the active site upon substrate binding to allow completion of the enzyme catalytic center, thanks to key residues that serve as hinges and anchoring points. In the substrate binding pocket, several highly conserved water molecules are coordinated by residues involved in substrate binding, comprising two water molecules very likely involved in the enzyme catalytic process. We also describe, by molecular dynamics simulations, how the transposition of the loop, besides providing the proximity of residues needed for catalysis, causes a general stabilization of the protein.

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