scholarly journals Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli

2020 ◽  
Vol 21 (12) ◽  
pp. 4212 ◽  
Author(s):  
Gracjana Klein ◽  
Pawel Wojtkiewicz ◽  
Daria Biernacka ◽  
Anna Stupak ◽  
Patrycja Gorzelak ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Chaperones ◽  
Active Site ◽  
Molecular Basis ◽  
Minimal Medium ◽  
Titration Calorimetry ◽  
Rich Medium ◽  
Wild Type ◽  
Ppiase Activity ◽  
Putative Active Site

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl cis/trans isomerases (PPIs). The Escherichia coli cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6ppi strains could be constructed only on minimal medium in the temperature range of 30–37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the ppiC gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC’s PPIase activity was shown to be required for interaction with AhpC.

scholarly journals Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

1990 ◽  
Vol 271 (2) ◽  
pp. 487-491 ◽  
Author(s):  
A Hädener ◽  
P R Alefounder ◽  
G J Hart ◽  
C Abell ◽  
A R Battersby
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Kinetic Studies ◽  
Enzyme Kinetic ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Over Expression ◽  
Putative Active Site ◽  
Lysine Residues

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5′-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.

scholarly journals Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners

2021 ◽  
Vol 22 (19) ◽  
pp. 10805
Author(s):  
José L. Neira ◽  
David Ortega-Alarcón ◽  
Bruno Rizzuti ◽  
Martina Palomino-Schätzlein ◽  
Adrián Velázquez-Campoy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Antibacterial Properties ◽  
Helical Structure ◽  
Titration Calorimetry ◽  
Its Sequence ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Residual Structure ◽  
Α Helix

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

Effect of ascorbate on oxygen uptake and growth of Escherichia coli B

1988 ◽  
Vol 34 (6) ◽  
pp. 822-824 ◽  
Author(s):  
Holly E. Richter ◽  
Jacek Switala ◽  
Peter C. Loewen
Keyword(s):  
Escherichia Coli ◽  
Oxygen Uptake ◽  
Electron Acceptor ◽  
Minimal Medium ◽  
Anaerobic Growth ◽  
Rich Medium ◽  
Terminal Electron Acceptor ◽  
Subsequent Change ◽  
Escherichia Coli B

The addition of ascorbate to aerobically growing cultures of Escherichia coli B caused only a short pause in growth and no subsequent change in the rate or extent of growth. The effect of ascorbate on oxygen uptake varied from inhibition in minimal medium to stimulation in rich medium. Cyanide-resistant growth and oxygen uptake were stimulated by ascorbate. Both the rate and extent of anaerobic growth were stimulated in proportion to the amount of ascorbate added when fumarate was the terminal electron acceptor. Ascorbate had no effect on any aspect of anaerobic growth in the absence of a terminal electron acceptor or in the presence of nitrate.

scholarly journals Disruption of aldA Influences the Developmental Process in Myxococcus xanthus

2000 ◽  
Vol 182 (2) ◽  
pp. 546-550 ◽  
Author(s):  
Mandy J. Ward ◽  
Helen Lew ◽  
David R. Zusman
Keyword(s):  
Gene Disruption ◽  
Minimal Medium ◽  
Developmental Process ◽  
Myxococcus Xanthus ◽  
Protein Sequences ◽  
Rich Medium ◽  
Wild Type ◽  
Rate Of Growth ◽  
Alanine Dehydrogenase ◽  
Developmental Conditions

ABSTRACT Previously, we identified a gene (aldA) fromMyxococcus xanthus, which we suggested encoded the enzyme alanine dehydrogenase on the basis of similarity to known Ald protein sequences (M. J. Ward, H. Lew, A. Treuner-Lange, and D. R. Zusman, J. Bacteriol. 180:5668–5675, 1998). In this study, we have confirmed that aldA does encode a functional alanine dehydrogenase, since it catalyzes the reversible conversion of alanine to pyruvate and ammonia. Whereas an aldA gene disruption mutation did not significantly influence the rate of growth or spreading on a rich medium, AldA was required for growth on a minimal medium containing l-alanine as the major source of carbon. Under developmental conditions, the aldA mutation caused delayed aggregation in both wild-type (DZ2) and FB (DZF1) strains. Poorly formed aggregates and reduced levels of spores were apparent in the DZ2 aldA mutant, even after prolonged development.

scholarly journals Medium Plays a Role in Determining Expression of acrB, marA, and soxS in Escherichia coli

2006 ◽  
Vol 50 (3) ◽  
pp. 1071-1074 ◽  
Author(s):  
Andrew M. Bailey ◽  
Mark A. Webber ◽  
Laura J. V. Piddock
Keyword(s):  
Escherichia Coli ◽  
Minimal Medium ◽  
Rich Medium ◽  
Growth Phases ◽  
Minimal Media ◽  
Logarithmic Growth

ABSTRACT Analysis of expression of acrB, marA, and soxS in rich and minimal media, at early and late logarithmic growth phases, showed that acrB had increased expression in minimal medium compared to rich medium, but expression decreased dose dependently upon exposure to ciprofloxacin.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue

2002 ◽  
Vol 365 (3) ◽  
pp. 809-816 ◽  
Author(s):  
Colin G. SAYSELL ◽  
Winston S. TAMBYRAJAH ◽  
Jeremy M. MURRAY ◽  
Carrie M. WILMOT ◽  
Simon E.V. PHILLIPS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Amine Oxidase ◽  
Reaction Pathway ◽  
Strong Support ◽  
Reversible Inhibitor ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Binding Data ◽  
Copper Amine

Copper amine oxidases are homodimeric enzymes containing one Cu2+ ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5–9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (Kb) against pH is bell-shaped, indicating two pKas of 5.8 and ∼8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the Kb versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

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