Do Thiol Amino Acids Inhibit Threonine Deaminase Activity in E. coli? (Letter to the Editor)

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

International Journal of Molecular Sciences ◽

10.3390/ijms20184416 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4416 ◽

Cited By ~ 2

Author(s):

Lara Console ◽

Maria Tolomeo ◽

Matilde Colella ◽

Maria Barile ◽

Cesare Indiveri

Keyword(s):

Cardiovascular Disease ◽

Amino Acids ◽

Risk Factor ◽

Biologically Active ◽

Suitable Model ◽

Native Protein ◽

E Coli ◽

Few Data ◽

Riboflavin Transport

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

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The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽

10.3390/catal11080926 ◽

2021 ◽

Vol 11 (8) ◽

pp. 926

Author(s):

Maria C. Martins ◽

Susana F. Fernandes ◽

Bruno A. Salgueiro ◽

Jéssica C. Soares ◽

Célia V. Romão ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Domain ◽

Reductase Activity ◽

Conserved Residues ◽

Mutant Proteins ◽

E Coli ◽

C Terminus ◽

Bona Fide ◽

Oxygen Reductase ◽

Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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Synthesis and Antibacterial Potency of 4-Methyl-2,7-dioxo-1,2,3,4,7,10- hexahydropyrido[2,3-ƒ]quinoxaline-8-carboxylic acid, Selected [a]-Fused Heterocyles and Acyclic Precursors

Zeitschrift für Naturforschung B ◽

10.1515/znb-2007-1115 ◽

2007 ◽

Vol 62 (11) ◽

pp. 1453-1458 ◽

Cited By ~ 8

Author(s):

Yusuf M. Al-Hiari ◽

Ali M. Qaisia ◽

Mohammad Y.Abu Shuheil ◽

Mustafa M. El-Abadelah ◽

Wolfgang Voelter

Keyword(s):

Amino Acids ◽

Carboxylic Acid ◽

Antibacterial Activities ◽

Sodium Dithionite ◽

E Coli ◽

Pipecolinic Acid

The reaction of 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7) with each of sarcosine and (±)-pipecolinic acid afforded the corresponding N-(4- oxoquinolin-7-yl)-α-amino acids 8 and 9. Reductive lactamization of the latter with sodium dithionite gave hexahydropyrido[2,3- f ]quinoxaline (10) and octahydrodipyrido[1,2-a : 2,3- f ]quinoxaline (11) derivatives, respectively. Compounds 8 - 11 and their homologs 1 - 6, accessible from (S)-proline, (2S, 4R)-4-hydroxyproline and (S)-tetrahydroisoquinoline-3-carboxylic acid exhibit good to excellent antibacterial activities against E. coli and S. aureus.

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ChemInform Abstract: Preparation of α-Deuterated L-Amino Acids with the Use of E. coli B/1t7-A Cells Containing Tryptophanase.

ChemInform ◽

10.1002/chin.199209277 ◽

2010 ◽

Vol 23 (9) ◽

pp. no-no

Author(s):

N. G. FALEEV ◽

S. B. RUVINOV ◽

M. B. SAPOROVSKAYA ◽

V. M. BELIKOV ◽

L. N. ZAKOMYRDINA ◽

...

Keyword(s):

Amino Acids ◽

E Coli ◽

A Cells

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Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽

10.1210/endo.138.2.4920 ◽

1997 ◽

Vol 138 (2) ◽

pp. 588-593 ◽

Cited By ~ 8

Author(s):

Y. Bobovnikova ◽

P. N. Graves ◽

H. Vlase ◽

T. F. Davies

Keyword(s):

Amino Acids ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Thioredoxin Reductase ◽

Biologically Active ◽

Receptor Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Fusion Partner ◽

E Coli ◽

Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

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Determination of amino acids misincorporation in recombinant protein by mass spectrometry

Bangladesh Journal of Animal Science ◽

10.3329/bjas.v42i1.15760 ◽

2013 ◽

Vol 42 (1) ◽

pp. 11-19 ◽

Cited By ~ 1

Author(s):

MZ Alam ◽

L Regioneiri ◽

MAS Santos

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Recombinant Protein ◽

Gene Fusion ◽

Aminoglycoside Antibiotic ◽

Protein Functionality ◽

Lacz Gene ◽

Host Organism ◽

E Coli

The synthesis of protein according to genetic code of a gene determines the basis of life and a stable proteome is necessary for cell homeostatis. However, errors occur naturally during translation of protein from its mRNA, which varies from 10-3 to 10-4 per codon. These errors are more frequent in recombinant protein overexpressed in heterologous hosts and affect protein functionality. The increasing amount of nonfunctional protein is often related to mistranslation of a gene under stress. In the present study, Saccharomyces cerevisiae as a host organism to overexpress E. coli lacZ gene fusion with GST to quantify misincorporation of amino acid in GST-? galactosidase recombinant protein. The yeast was treated with various stressors such as ethanol, chromium (CrO3), and aminoglycoside antibiotic - geneticin (G418) to induce protein aggregation. The misincorporation of amino acids was studied in soluble protein fractions by mass-spectrometry to determine how much misincorporation occur. We found that under experimental stress conditions the misincorporation of amino acids ranges from 5.6 ×10-3 to 8 × 10-3, which represents 60-80 fold higher than reported level. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15760 Bang. J. Anim. Sci. 2013. 42 (1): 11-19

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Computation of condition-dependent proteome allocation reveals variability in the macro and micro nutrient requirements for growth

10.1101/2020.03.23.003236 ◽

2020 ◽

Cited By ~ 1

Author(s):

Colton J. Lloyd ◽

Jonathan Monk ◽

Laurence Yang ◽

Ali Ebrahim ◽

Bernhard O. Palsson

Keyword(s):

Amino Acids ◽

Growth Conditions ◽

Anaerobic Growth ◽

Metabolic Phenotype ◽

E Coli ◽

Scale Models ◽

Protein Components ◽

K 12 ◽

Genome Scale ◽

Prosthetic Groups

AbstractSustaining a robust metabolic network requires a balanced and fully functioning proteome. In addition to amino acids, many enzymes require cofactors (coenzymes and engrafted prosthetic groups) to function properly. Extensively validated genome-scale models of metabolism and gene expression (ME-models) have the unique ability to compute an optimal proteome composition underlying a metabolic phenotype, including the provision of all required cofactors. Here we use the ME-model for Escherichia coli K-12 MG1655 to computationally examine how environmental conditions change the proteome and its accompanying cofactor usage. We found that: (1) The cofactor requirements computed by the ME model mostly agree with the standard biomass objective function used in models of metabolism alone (M models); (2) ME-model computations reveal non-intuitive variability in cofactor use under different growth conditions; (3) An analysis of ME-model predicted protein use in aerobic and anaerobic conditions suggests an enrichment in the use of prebiotic amino acids in the proteins used to sustain anaerobic growth (4) The ME-model could describe how limitation in key protein components affect the metabolic state of E. coli. Genome-scale models have thus reached a level of sophistication where they reveal intricate properties of functional proteomes and how they support different E. coli lifestyles.

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Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽

10.1099/mic.0.001087 ◽

2021 ◽

Vol 167 (9) ◽

Author(s):

Nirbhay Singh ◽

Anu Chauhan ◽

Ram Kumar ◽

Sudheer Kumar Singh

Keyword(s):

Amino Acids ◽

Mycobacterium Tuberculosis ◽

Type Species ◽

Low Ph ◽

Stress Conditions ◽

Starvation Stress ◽

Content Type ◽

E Coli ◽

Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

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