scholarly journals Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase

1999 ◽  
Vol 341 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Michael T. STUMPP ◽  
Ken MOTOHASHI ◽  
Toru HISABORI
Keyword(s):  
Active Site ◽  
High Efficiency ◽  
Expression System ◽  
Water Soluble ◽  
Subsequent Reduction ◽  
Disulphide Bridge ◽  
Mutant Proteins ◽  
Apparent Affinity ◽  
Γ Subunit

The activity of the chloroplast H+-ATPase (CFoCF1) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the γ-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CFoCF1 (CF1) and includes two successive steps, namely the binding of Trx to CF1 and the subsequent reduction or oxidation of CF1. To study this process thoroughly, a new expression system for spinach Trx-f and Trx-m was designed. In the presence of dithiothreitol (DTT) both forms of the expressed Trx could reduce the disulphide bridge on the γ-subunit of CF1 and thus activate the ATPase. Trx mutants deficient in the internal, or both, cysteines of the active site were designed to study the details of the interaction. The Trx mutant proteins could still activate CF1-ATPase in the presence of DTT and they also increased the apparent affinity of CF1 for DTT. This implies that the binding of Trx to the CF1 γ-subunit induces a conformational change facilitating the reduction of the disulphide bridge, and partially explains the high efficiency of Trx as a reductant in vivo.

scholarly journals Functional Expression of a DNA-Topoisomerase IB fromCryptosporidium parvum

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
César Ordóñez ◽  
Javier Alfonso ◽  
Rafael Balaña-Fouce ◽  
David Ordóñez
Keyword(s):  
Expression System ◽  
Functional Expression ◽  
Structural Features ◽  
Water Soluble ◽  
Irreversible Inhibitor ◽  
Dna Topoisomerase ◽  
Yeast Expression System ◽  
Topoisomerase Ib ◽  
Single Polypeptide

Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB) in chromosome 7. Heterologous expression ofCpTopIBgene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast extracts was detected only whenCpTopIBORF was expressed in a yeast expression system showing time and protein dependence under steady state kinetic conditions. The susceptibility of CpTopIB-transformed yeast to the irreversible inhibitor camptothecin and its water-soluble derivatives (topotecan and SN-38) was assessed.

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.

scholarly journals Biosynthesis of Cyanobacterial Phycobiliproteins in Escherichia coli: Chromophorylation Efficiency and Specificity of All Bilin Lyases from Synechococcus sp. Strain PCC 7002

2010 ◽  
Vol 76 (9) ◽  
pp. 2729-2739 ◽  
Author(s):  
Avijit Biswas ◽  
Yasmin M. Vasquez ◽  
Tierna M. Dragomani ◽  
Monica L. Kronfel ◽  
Shervonda R. Williams ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Expression System ◽  
Water Soluble ◽  
Soluble Form ◽  
Beta Subunits ◽  
Lyase Activity ◽  
Synechococcus Sp ◽  
Tetrapyrrole Chromophores

ABSTRACT Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and α-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter−1 of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (αAP-B) and ApcF (β18). The N-terminal, allophycocyanin-like domain of ApcE (LCM 99) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of β-phycocyanin.

Highly efficient BODIPY-doped upconversion nanoparticles for deep-red luminescence bioimaging in vivo

2021 ◽  
Author(s):  
Ti Jia ◽  
Qiuhong Wang ◽  
Ming Xu ◽  
Wei Yuan ◽  
Wei Feng ◽  
...  
Keyword(s):  
High Efficiency ◽  
Upconversion Luminescence ◽  
Excitation Power ◽  
Water Soluble ◽  
Upconversion Nanoparticles ◽  
Highly Efficient ◽  
Red Luminescence

We demonstrate a 3,5-di(p-oxethyl)styryl conjugated BODIPY showing deep-red upconversion luminescence with a high efficiency of 16.6%. Furthermore, water-soluble BODIPY-doped upconversion nanoparticles with efficiency up to 6.9% under low excitation power...

scholarly journals Atomically precise silver clusterzymes protect mice from radiation damages

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jiao Guo ◽  
Haiyu Yang ◽  
Ya Liu ◽  
Wei Liu ◽  
Ruiying Zhao ◽  
...  
Keyword(s):  
Cell Viability ◽  
High Efficiency ◽  
Disease Diagnosis ◽  
The Body ◽  
Water Soluble ◽  
Whole Body ◽  
Dna Damages ◽  
Γ Ray

Abstract Background As we know, radiotherapy plays an irreplaceable role in the clinical management on solid tumors. However, due to the non-specific killing effects of ionizing radiation, normal tissues damages would be almost simultaneous inevitably. Therefore, ideal radioprotective agents with high efficiency and low toxicity are always desirable. In this work, atomically precise Ag14 clusterzymes were developed, and their applications in radioprotection were studied in vitro and in vivo for the first time. Methods The ultra-small glutathione supported Ag14 clusterzymes were synthesized by convenient sodium borohydride (NaBH4) reduction of thiolate-Ag (I) complexes and then they were purified by desalting columns. The enzyme-like activity and antioxidant capacity of Ag14 clusterzymes have been tested by various commercial kits, salicylic acid method and electron spin resonance (ESR). Next, they were incubated with L929 cells to evaluate whether they could increase cell viability after γ-ray irradiation. And then Ag14 clusterzymes were intravenously injected into C57 mice before 7 Gy whole-body γ-ray irradiation to evaluate the radioprotection effects in vivo. At last, the in vivo toxicities of Ag14 clusterzymes were evaluated through biodistribution test, hematological details, serum biochemical indexes and histological test in female Balb/c mice with intravenous injection of Ag14 clusterzymes. Results Our studies suggested atomically precise Ag14 clusterzymes were potential radioprotectants. Ag14 clusterzymes exhibited unique superoxide dismutase (SOD)-like activity, strong anti-oxidative abilities, especially on •OH scavenging. The Ag14 clusterzymes could effectively improve cell viability through eliminating ROS and prevent DNA damages in cells dealt with γ-ray irradiation. In vivo experiments showed that Ag14 clusterzymes could improve the irradiated mice survival rate by protecting hematological systems and repairing tissue oxidative stress damage generated by γ-ray irradiation. In addition, bio-distribution and toxicological experiments demonstrated that the ultrasmall Ag14 clusterzymes could be excreted quickly from the body by renal clearance and negligible toxicological responses were observed in mice up to 30 days. Conclusion In summary, atomically precise, ultrasmall and water soluble Ag14 clusterzymes with SOD-like activity were successfully developed and proved to be effective both in vitro and in vivo for radioprotection. Furthermore, with atomically precise molecular structure, Ag14 clusterzymes, on aspect of the catalytic and optical properties, may be improved by structure optimization on atom-scale level for other applications in disease diagnosis and treatment. Graphical Abstract

scholarly journals Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue

1990 ◽  
Vol 269 (2) ◽  
pp. 443-450 ◽  
Author(s):  
G C Russell ◽  
J R Guest
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Histidine Residue ◽  
Complex Activity ◽  
Putative Active Site ◽  
Active Site Histidine

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.

scholarly journals Substitutions in an Active Site Loop ofEscherichia coliIscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesisin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 19551-19558 ◽  
Author(s):  
Charles T. Lauhon ◽  
Elizabeth Skovran ◽  
Hugo D. Urbina ◽  
Diana M. Downs ◽  
Larry E. Vickery
Keyword(s):  
Active Site ◽  
Cluster Formation ◽  
Wild Type ◽  
Cysteine Desulfurase ◽  
Mutant Proteins ◽  
Active Site Cysteine ◽  
Sulfur Transfer ◽  
Amino Acid Region

IscS catalyzes the fragmentation ofl-cysteine tol-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. InEscherichia coliIscS, the active site cysteine Cys328resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys328identified additional residues important for IscS functionin vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesisin vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s2C and ms2i6A) yet showed wild type levels of Fe-S-independent thionucleosides (s4U and mnm5s2U) that require persulfide formation and transfer.In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesisin vivowithout detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

scholarly journals Efficacy of Water-Soluble Pearl Powder Components Extracted by a CO2 Supercritical Extraction System in Promoting Wound Healing

Materials ◽  
2021 ◽  
Vol 14 (16) ◽  
pp. 4458
Author(s):  
Minting Liu ◽  
Junjun Tao ◽  
Hongchen Guo ◽  
Liang Tang ◽  
Guorui Zhang ◽  
...  
Keyword(s):  
Wound Healing ◽  
High Efficiency ◽  
Extraction Process ◽  
Water Soluble ◽  
Biologically Active ◽  
Supercritical Extraction ◽  
Extraction System ◽  
Collagen Formation

Pearl powder is a biologically active substance that is widely used in traditional medicine, skin repair and maintenance. The traditional industrial extraction processes of pearl powder are mainly based on water, acid or enzyme extraction methods, all of which have their own drawbacks. In this study, we propose a new extraction process for these active ingredients, specifically, water-soluble components of pearl powder extracted by a CO2 supercritical extraction system (SFE), followed by the extraction efficiency evaluation. A wound-healing activity was evaluated in vitro and in vivo. This demonstrated that the supercritical extraction technique showed high efficiency as measured by the total protein percentage. The extracts exhibited cell proliferation and migration-promoting activity, in addition to improving collagen formation and healing efficiency in vivo. In brief, this study proposes a novel extraction process for pearl powder, and the extracts were also explored for wound-healing bioactivity, demonstrating the potential in wound healing.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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