Faculty Opinions recommendation of Barriers to Hydride Transfer in Wild Type and Mutant Dihydrofolate Reductase from E. coli.

2003 ◽  
Author(s):  
Arieh Warshel
Keyword(s):  
Dihydrofolate Reductase ◽  
Hydride Transfer ◽  
Wild Type ◽  
E Coli

scholarly journals Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima

2003 ◽  
Vol 374 (2) ◽  
pp. 529-535 ◽  
Author(s):  
Giovanni MAGLIA ◽  
Masood H. JAVED ◽  
Rudolf K. ALLEMANN
Keyword(s):  
Steady State ◽  
Dihydrofolate Reductase ◽  
Tertiary Structure ◽  
Thermotoga Maritima ◽  
Hydride Transfer ◽  
Kinetic Scheme ◽  
Transfer Rates ◽  
E Coli ◽  
Steady State Kinetics ◽  
Rate Limiting

DHFR (dihydrofolate reductase) catalyses the metabolically important reduction of 7,8-dihydrofolate by NADPH. DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR), which shares similarity with DHFR from Escherichia coli, has previously been characterized structurally. Its tertiary structure is similar to that of DHFR from E. coli but it is the only DHFR characterized so far that relies on dimerization for stability. The midpoint of the thermal unfolding of TmDHFR was at approx. 83 °C, which was 30 °C higher than the melting temperature of DHFR from E. coli. The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy. The rate constant for hydride transfer was found to depend strongly on the temperature and the pH of the solution. Hydride transfer was slow (0.14 s−1 at 25 °C) and at least partially rate limiting at low temperatures but increased dramatically with temperature. At 80 °C the hydride-transfer rate of TmDHFR was 20 times lower than that observed for the E. coli enzyme at its physiological temperature. Hydride transfer depended on ionization of a single group in the active site with a pKa of 6.0. While at 30 °C, turnover of substrate by TmDHFR was almost two orders of magnitude slower than by DHFR from E. coli; the steady-state rates of the two enzymes differed only 8-fold at their respective working temperatures.

High-resolution gold labeling

1993 ◽  
Vol 51 ◽  
pp. 330-331
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya ◽  
Kyra Carbone ◽  
Martha Simon ◽  
Beth Lin ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Polypeptide Chain ◽  
External Surface ◽  
Gold Cluster ◽  
Macromolecular Complex ◽  
Gold Compound ◽  
Central Cavity ◽  
Chain Folding ◽  
E Coli ◽  
Chaperonin Groel

A recently developed 1.4 nm gold cluster has been found to be useful in labeling macromolecular sites to 1-3 nm resolution. The gold compound is organically derivatized to contain a monofunctional arm for covalent linking to biomolecules. This may be used to mark a specific site on a structure, or to first label a component and then reassemble a multicomponent macromolecular complex. Two examples are given here: the chaperonin groEL and ribosomes.Chaperonins are essential oligomeric complexes that mediate nascent polypeptide chain folding to produce active proteins. The E. coli chaperonin, groEL, has two stacked rings with a central hole ∽6 nm in diameter. The protein dihydrofolate reductase (DHFR) is a small protein that has been used in chain folding experiments, and serves as a model substrate for groEL. By labeling the DHFR with gold, its position with respect to the groEL complex can be followed. In particular, it was sought to determine if DHFR refolds on the external surface of the groEL complex, or whether it interacts in the central cavity.

The Effect of Mianserin on Lifespan of Caenorhabditis elegans is Abolished by Glucose

2021 ◽  
Vol 13 ◽  
Author(s):  
Abdullah Almotayri ◽  
Jency Thomas ◽  
Mihiri Munasinghe ◽  
Markandeya Jois
Keyword(s):  
Caenorhabditis Elegans ◽  
Scaffolding Protein ◽  
Lifespan Extension ◽  
Wild Type ◽  
E Coli ◽  
C Elegans ◽  
Risk Of Death ◽  
Increased Risk ◽  
Antidepressant Use ◽  
Extension Effect

Background: The antidepressant mianserin has been shown to extend the lifespan of Caenorhabditis elegans (C. elegans), a well-established model organism used in aging research. The extension of lifespan in C. elegans was shown to be dependent on increased expression of the scaffolding protein (ANK3/unc-44). In contrast, antidepressant use in humans is associated with an increased risk of death. The C. elegans in the laboratory are fed Escherichia coli (E. coli), a diet high in protein and low in carbohydrate, whereas a typical human diet is high in carbohydrates. We hypothesized that dietary carbohydrates might mitigate the lifespan-extension effect of mianserin. Objective: To investigate the effect of glucose added to the diet of C. elegans on the lifespan-extension effect of mianserin. Methods: Wild-type Bristol N2 and ANK3/unc-44 inactivating mutants were cultured on agar plates containing nematode growth medium and fed E. coli. Treatment groups included (C) control, (M50) 50 μM mianserin, (G) 73 mM glucose, and (M50G) 50 μM mianserin and 73 mM glucose. Lifespan was determined by monitoring the worms until they died. Statistical analysis was performed using the Kaplan-Meier version of the log-rank test. Results: Mianserin treatment resulted in a 12% increase in lifespan (P<0.05) of wild-type Bristol N2 worms but reduced lifespan by 6% in ANK3/unc-44 mutants, consistent with previous research. The addition of glucose to the diet reduced the lifespan of both strains of worms and abolished the lifespan-extension by mianserin. Conclusion: The addition of glucose to the diet of C. elegans abolishes the lifespan-extension effects of mianserin.

New insights in gut-liver axis in wild-type murine imiquimod-induced lupus

Lupus ◽  
2021 ◽  
Vol 30 (6) ◽  
pp. 926-936
Author(s):  
Georges Maalouly ◽  
Joelle Hajal ◽  
Charbel Noujeim ◽  
Michel Choueiry ◽  
Hussein Nassereddine ◽  
...  
Keyword(s):  
Tight Junction ◽  
Fecal Calprotectin ◽  
Liver Inflammation ◽  
Tight Junction Proteins ◽  
Wild Type ◽  
Imiquimod Cream ◽  
E Coli ◽  
Intestinal Pathology ◽  
Nfκb Pathway ◽  
Junction Proteins

Background Intestinal and hepatic manifestations of lupus seem to be underestimated in comparison to other major organ lesions. Although recent data point to gut-liver axis involvement in lupus, gut permeability dysfunction and liver inflammation need to be more investigated. Objective This study aims to assess fecal calprotectin, intestinal tight junction proteins and liver inflammation pathway in wild-type murine imiquimod- induced lupus. Methods C57BL/6 mice were topically treated on their right ears with 1.25 mg of 5% imiquimod cream, three times per week for six weeks. Fecal calprotectin was collected at day 0, 22 and 45. Renal, liver and intestinal pathology, as well as inflammatory markers, intestinal tight junction proteins, and E. coli protein in liver were assessed at sacrifice. Results At six weeks, lupus nephritis was confirmed on histopathology and NGAL and KIM-1 expression. Calprotectin rise started at day 22 and persists at day 45. Protein expression of Claudine, ZO-1 and occludin was significantly decreased. E. coli protein was significantly increased in liver with necro-inflammation and increased TLR4, TLR7, and pNFκB/NFκB liver expression. Conclusion This study is the first to demonstrate early fecal calprotectin increase and liver activation of TLR4- NFκB pathway in wild-type murine imiquimod-induced lupus.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced &gt;250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

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.

Arabidopsis AtCNGC10 rescues potassium channel mutants of E. coli, yeast and Arabidopsis and is regulated by calcium/calmodulin and cyclic GMP in E. coli

2005 ◽  
Vol 32 (7) ◽  
pp. 643 ◽  
Author(s):  
Xinli Li ◽  
Tamás Borsics ◽  
H. Michael Harrington ◽  
David A. Christopher
Keyword(s):  
Channel Blocker ◽  
K Channel ◽  
Structure Characteristic ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
E Coli ◽  
Diverse Species ◽  
The Family ◽  
Low K

We have isolated and characterised AtCNGC10, one of the 20 members of the family of cyclic nucleotide (CN)-gated and calmodulin (CaM)-regulated channels (CNGCs) from Arabidopsis thaliana (L.) Heynh. AtCNGC10 bound CaM in a C-terminal subregion that contains a basic amphiphillic structure characteristic of CaM-binding proteins and that also overlaps with the predicted CN-binding domain. AtCNGC10 is insensitive to the broad-range K+ channel blocker, tetraethylammonium, and lacks a typical K+-signature motif. However, AtCNGC10 complemented K+ channel uptake mutants of Escherichia coli (LB650), yeast (Saccharomyces cerevisiae CY162) and Arabidopsis (akt1-1). Sense 35S-AtCNGC10 transformed into the Arabidopsis akt1-1 mutant, grew 1.7-fold better on K+-limited medium relative to the vector control. Coexpression of CaM and AtCNGC10 in E. coli showed that Ca2+ / CaM inhibited cell growth by 40%, while cGMP reversed the inhibition by Ca2+ / CaM, in a AtCNGC10-dependent manner. AtCNGC10 did not confer tolerance to Cs+ in E. coli, however, it confers tolerance to toxic levels of Na+ and Cs+ in the yeast K+ uptake mutant grown on low K+ medium. Antisense AtCNGC10 plants had 50% less potassium than wild type Columbia. Taken together, the studies from three evolutionarily diverse species demonstrated a role for the CaM-binding channel, AtCNGC10, in mediating the uptake of K+ in plants.

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