Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structure of Escherichia coli tryptophanase purified from an alkaline-stressed bacterial culture.

ABSTRACT: Tryptophanase is a bacterial enzyme involved in the degradation of tryptophan to indole, pyruvate and ammonia, which are compounds that are essential for bacterial survival. Tryptophanase is often overexpressed in stressed cultures. Large amounts of endogenous tryptophanase were purified from Escherichia coli BL21 strain overexpressing another recombinant protein. Tryptophanase was crystallized in space group P6522 in the apo form without pyridoxal 5'-phosphate bound in the active site.

SUBMITTER: Rety S

PROVIDER: S-EPMC4631586 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Structure of Escherichia coli tryptophanase purified from an alkaline-stressed bacterial culture.

Rety Stephane S Deschamps Patrick P Leulliot Nicolas N

Acta crystallographica. Section F, Structural biology communications 20151023 Pt 11

Tryptophanase is a bacterial enzyme involved in the degradation of tryptophan to indole, pyruvate and ammonia, which are compounds that are essential for bacterial survival. Tryptophanase is often overexpressed in stressed cultures. Large amounts of endogenous tryptophanase were purified from Escherichia coli BL21 strain overexpressing another recombinant protein. Tryptophanase was crystallized in space group P6522 in the apo form without pyridoxal 5'-phosphate bound in the active site. ...[more]

PMID: 26527264

Similar Datasets

Structures of Escherichia coli tryptophanase in holo and 'semi-holo' forms.

Project description:Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9?Å resolution. The second crystal form diffracted to 3.2?Å resolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two sulfate ions bound in the vicinity of the active site. The conformation of all holo subunits is the same in both crystal forms. The structures suggest that Trpase is flexible in the apo form. Its conformation partially closes upon binding of PLP. The closed conformation might correspond to the enzyme in its active state with both cofactor and substrate bound in a similar way as in tyrosine phenol-lyase.

| S-EPMC4356303 | biostudies-literature

Cytoplasmic protein mobility in osmotically stressed Escherichia coli.

Project description:Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.

| S-EPMC2612437 | biostudies-literature

Cell-free protein synthesis from non-growing, stressed Escherichia coli.

Project description:Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.

| S-EPMC5705671 | biostudies-literature

The antibiotics potentiator bicarbonate causes upregulation of tryptophanase and iron acquisition proteins in Escherichia coli

Project description:We have reported that bicarbonate (NaHCO3) potentiates the activity of aminoglycosides in Escherichia coli, but the action mechanism was not identified. To eventually understand how NaHCO3 can potentiate antibiotics, we thought that a rational first step was to examine the effect of NaHCO3 separately and to inspect initial gene expression changes triggered by it. In this work we started by confirming that NaHCO3 can reduce the number of viable E. coli bacteria. We then investigated, via RNAseq, gene expression changes induced by NaHCO3. There were upregulated and downregulated genes, among the top upregulated genes (~10-fold increase in expression) was tnaA, the gene encoding tryptophanase (TnaA), the enzyme that degrades tryptophan to indole. Considering that higher expression of tnaA likely led to increases in indole, we tested the effect of indole and found both growth inhibition and synergy with NaHCO3. We suggest that indole may participate in growth inhibition of E. coli. The RNAseq analysis also revealed upregulation (≥4-fold) of genes encoding proteins for the acquisition of iron and downregulation (≥16-fold) of genes encoding iron-sulfur-holding proteins, hence NaHCO3 apparently triggered also an iron deficit response. We suggest that iron deficiency may also be involved in growth inhibition by NaHCO3.

2018-11-01 | GSE110493 | GEO

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase.

Project description:Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62-242 milliunits/mg and phosphate ester hydrolysis with specific activities of 41-61 units/mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H2. Thus, BAP is a unique Pt-dependent, H2-evolving hydrogenase. This reaction is unprecedented in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.

| S-EPMC419532 | biostudies-literature

Production of Indigo by Recombinant <i>Escherichia coli</i> with Expression of Monooxygenase, Tryptophanase, and Molecular Chaperone.

Project description:Indigo is an important pigment widely used in industries of food, cosmetics, and textile. In this work, the styrene monooxygenase StyAB from Pseudomonas putida was co-expressed with the tryptophanase TnaA and the chaperone groES-groEL in Escherichia coli for indigo production. Over-expression of the gene styAB endowed the recombinant E. coli AB with the capacity of indigo biosynthesis from indole and tryptophan. Tryptophan fermentation in E. coli AB generated about five times more indigo than that from indole, and the maximum 530 mg/L of indigo was obtained from 1.2 mg/mL of tryptophan. The gene TnaA was then co-expressed with styAB, and the tryptophanase activity significantly increased in the recombinant E. coli ABT. However, TnaA expression led to a decrease in the activity of StyAB and indigo yield in E. coli ABT. Furthermore, the plasmid pGro7 harboring groES-groEL was introduced into E. coli AB, which obviously promoted the activity of StyAB and accelerated indigo biosynthesis in the recombinant E. coli ABP. In addition, the maximum yield of indigo was further increased to 550 mg/L from 1.2 mg/mL of tryptophan in E. coli ABP. The genetic manipulation strategy proposed in this work could provide new insights into construction of indigo biosynthesis cell factory for industrial production.

| S-EPMC9320885 | biostudies-literature

Escherichia coli biofilms vs planktonic culture

Project description:Transcription profile of Escherichia coli cells in biofilms under static batch culture was compared to that of E. coli cells in planktonic cultures. Both E. coli biofilm and planktonic cultures were cultivated for 18 h in 10% Luria-Bertani broth at room temperature (20 degree Celsius). Biofilms were grown in static batch culture in petri dishes. Both planktonic culture and biofilms were homogenized and run through a separated protocol.

2013-12-30 | GSE24914 | GEO

Distinct structure and activity recoveries reveal differences in metal binding between mammalian and Escherichia coli alkaline phosphatases.

Project description:The amino acids involved in the coordination of two Zn2+ ions and one Mg2+ ion in the active site are well conserved from EAP (Escherichia coli alkaline phosphatase) to BIAP (bovine intestinal alkaline phosphatase), whereas most of their surrounding residues are different. To verify the consequences of this heterology on their specific activities, we compared the activity and structure recoveries of the metal-free forms (apo) of EAP and of BIAP. In the present study, we found that although the sensitivities of EAP and BIAP to ions remained similar, significant differences in dimeric structure stability of apo-enzymes were observed between EAP and BIAP, as well as in the kinetics of their activity and secondary structure recoveries. After mild chelation inactive apo-EAP was monomeric under mild denaturing conditions, whereas inactive apo-BIAP remained dimeric, indicating that the monomer-monomer contact was stronger in the mammalian enzyme. Dimeric apo-EAP (0.45 microM, corresponding to 4 units/ml) recovered approx. 80% of its initial activity after 3 min incubation in an optimal recovery medium containing 5 microM Zn2+ and 5 mM Mg2+, whereas dimeric apo-BIAP (0.016 microM, corresponding to 4 units/ml) recovered 80% of its native activity after 6 h incubation in an optimal recovery medium containing 0.5 microM Zn2+ and 5 mM Mg2+. Small and different secondary structure changes were also observed during activity recoveries of apo-BIAP and apo-EAP, which were not in parallel with the activity recoveries, suggesting that distinct and subtle structural changes are required for their optimal activity recoveries.

| S-EPMC1316277 | biostudies-literature

Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli.

Project description:1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).

| S-EPMC1176966 | biostudies-other

Escherichia coli biofilm: mono-species culture vs. mixed-species culture

Project description:Transcription profile of Escherichia coli cells in mono-species pure biofilms was compared to that of E. coli cells in E. coli-Stenotrophomonas maltophilia dual-species biofilms. E. coli cells were separated from dual-species biofilms before total RNA extraction to eliminate possible cross hybridization from S. maltophilia transcripts. The separation method was developed by combining the use of reagent RNAlater and immuno-magnetic separation. Pure E. coli biofilms were processed with the same separation protocol before RNA extraction.

2013-12-30 | GSE24912 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data