Glutamic acid-141: a heme ‘bodyguard’ in anionic tobacco peroxidase

2007 ◽  
Vol 388 (4) ◽  
pp. 373-380 ◽  
Author(s):  
Dmitri M. Hushpulian ◽  
Andrew A. Poloznikov ◽  
Pavel A. Savitski ◽  
Alexandra M. Rozhkova ◽  
Tatyana A. Chubar ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Active Center ◽  
Structural Alignment ◽  
Wild Type ◽  
E Coli ◽  
Anionic Peroxidase ◽  
Glutamic Acid Residue ◽  
Plant Peroxidases ◽  
Tobacco Peroxidase

Abstract The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it ‘guards’ heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes

1990 ◽  
Vol 10 (12) ◽  
pp. 6257-6263
Author(s):  
A Frankel ◽  
P Welsh ◽  
J Richardson ◽  
J D Robertus
Keyword(s):  
Enzyme Activity ◽  
Glutamic Acid ◽  
Structural Information ◽  
Wild Type ◽  
Toxin A ◽  
Mutant Proteins ◽  
Ricin Toxin ◽  
Enzymatic Inactivation ◽  
A Chain

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

scholarly journals Role of CheB and CheR in the Complex Chemotactic and Aerotactic Pathway of Azospirillum brasilense

2006 ◽  
Vol 188 (13) ◽  
pp. 4759-4768 ◽  
Author(s):  
Bonnie B. Stephens ◽  
Star N. Loar ◽  
Gladys Alexandre
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Azospirillum Brasilense ◽  
Model Organism ◽  
Spatial Gradient ◽  
Wild Type ◽  
Temporal Gradient ◽  
E Coli ◽  
Significant Difference

ABSTRACT It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.

scholarly journals Role of Quorum Sensing and Antimicrobial Component Production by Serratia plymuthica in Formation of Biofilms, Including Mixed Biofilms with Escherichia coli

2006 ◽  
Vol 72 (11) ◽  
pp. 7294-7300 ◽  
Author(s):  
Pieter Moons ◽  
Rob Van Houdt ◽  
Abram Aertsen ◽  
Kristof Vanoirbeek ◽  
Yves Engelborghs ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Broth Culture ◽  
Confocal Laser ◽  
Wild Type ◽  
Serratia Plymuthica ◽  
E Coli

ABSTRACT We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.

scholarly journals Overexpression of Mannitol-1-Phosphate Dehydrogenase Increases Mannitol Accumulation and Adds Protection Against Chilling Injury in Petunia

2005 ◽  
Vol 130 (4) ◽  
pp. 605-610 ◽  
Author(s):  
Yu-Jen Chiang ◽  
C. Stushnoff ◽  
A.E. McSay ◽  
M.L. Jones ◽  
H.J. Bohnert
Keyword(s):  
Chilling Stress ◽  
Chilling Injury ◽  
Dry Weight ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Horticultural Crops ◽  
Transgenic Lines ◽  
And Control

Petunia ×hybrida (Hook) Vilm. cv. Mitchell was transformed with an E. coli gene encoding mannitol-1-phosphate dehydrogenase (mtlD). Four plant lines that grew on kanamycin and contained the mtlD transgene were identified. Two of these lines contained high levels of mannitol [high-mannitol lines M3 and M8; mean mannitol = 3.39 μmol·g-1 dry weight (DW)] compared to nontransformed wild-type plants (0.86 μmol·g-1 DW), while two lines had mannitol levels similar to wild-type plants (low-mannitol lines M2 and M9; mean mannitol = 1.05 μmol·g-1 DW). Transgenic and control plants were subjected to chilling stress (3 ± 0.5 °C day/0 ± 0.5 °C night, 12-hour photoperiod and 75% relative humidity) to evaluate the role of mannitol in chilling tolerance. Based upon foliage symptoms and membrane leakage after a 3-week chilling treatment, the high-mannitol containing lines, M3 and M8, were more tolerant of chilling stress than the low-mannitol containing transgenic lines, M2 and M9, and wild-type. Under nonchilling conditions mannitol was the only carbohydrate that differed among transgenic lines, but all carbohydrates were present. When subjected to chilling stress, mannitol levels dropped by 75%, sucrose by 52%, and inositol by 54% in the low-mannitol lines (M2 and M9). In M3 and M8, the high-mannitol lines, mannitol levels decreased by 36%, sucrose by 25%, and inositol by 56%, respectively. Raffinose increased 2- to 3-fold in all lines following exposure to low-temperature chilling stress. In the higher mannitol lines only 0.04% to 0.06% of the total osmotic potential generated from all solutes could be attributed to mannitol, thus its action is more like that of an osmoprotectant rather than an osmoregulator. This study demonstrates that metabolic engineering of osmoprotectant synthesis pathways can be used to improve stress tolerance in horticultural crops.

scholarly journals Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

1989 ◽  
Vol 9 (11) ◽  
pp. 5012-5021 ◽  
Author(s):  
D Schlossman ◽  
D Withers ◽  
P Welsh ◽  
A Alexander ◽  
J Robertus ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Toxin A ◽  
Mutant Proteins ◽  
Ricin Toxin ◽  
Glutamic Acid Residue ◽  
Enzymatic Inactivation ◽  
Sequence Encoding ◽  
A Chain ◽  
Beta Galactosidase

The gene for the A chain of ricin toxin was fused to a beta-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.

scholarly journals Evidence that SpoIVFB Is a Novel Type of Membrane Metalloprotease Governing Intercompartmental Communication duringBacillus subtilis Sporulation

2000 ◽  
Vol 182 (11) ◽  
pp. 3305-3309 ◽  
Author(s):  
Yuen-Tsu Nicco Yu ◽  
Lee Kroos
Keyword(s):  
Bacillus Subtilis ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Single Amino Acid ◽  
Wild Type ◽  
Cell Compartment ◽  
Transmembrane Segments ◽  
Glutamic Acid Residue ◽  
Isoleucine Residue ◽  
Hexxh Motif

ABSTRACT Processing of pro-ςK in the mother cell compartment of sporulating Bacillus subtilis involves SpoIVFB and is governed by a signal from the forespore. SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments. Several conservative single amino acid changes in the HEXXH motif abolished function. However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function. Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence. Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases. A conservative change in this region of SpoIVFB abolished function. Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.

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