The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N.

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Single Amino Acid Substitutions at Specific Positions of the Heptad Repeat Sequence of Piscidin-1 Yielded Novel Analogs That Show Low Cytotoxicity andIn VitroandIn VivoAntiendotoxin Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02341-15 ◽

2016 ◽

Vol 60 (6) ◽

pp. 3687-3699 ◽

Cited By ~ 15

Author(s):

Amit Kumar ◽

Amit Kumar Tripathi ◽

Manoj Kathuria ◽

Sonal Shree ◽

Jitendra Kumar Tripathi ◽

...

Keyword(s):

Body Weight ◽

Biological Activity ◽

Amino Acid ◽

Mammalian Cells ◽

Functional Characterization ◽

Repeat Sequence ◽

Heptad Repeat ◽

Single Amino Acid ◽

Cytotoxic Activities ◽

Isoleucine Residue

Piscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 heptad repeat.

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Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

10.1101/789503 ◽

2019 ◽

Author(s):

Haisu Shi ◽

Jinlong Tian ◽

Chen Wu ◽

Mo Li ◽

Feiyu An ◽

...

Keyword(s):

Amino Acids ◽

Catalytic Activity ◽

Fatty Acid ◽

Amino Acid ◽

Molecular Mechanism ◽

Active Sites ◽

Transmembrane Domain ◽

Fatty Acid Desaturase ◽

Domain Swapping ◽

The Third

AbstractCheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

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Functional characterization of the type I toxin Lpt from Lactobacillus rhamnosus by fluorescence and atomic force microscopy

Scientific Reports ◽

10.1038/s41598-019-51523-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Stefano Maggi ◽

Korotoum Yabre ◽

Alberto Ferrari ◽

Camilla Lazzi ◽

Mitsuoki Kawano ◽

...

Keyword(s):

Amino Acid ◽

Surface Defects ◽

Fluorescent Protein ◽

Functional Characterization ◽

Lactobacillus Rhamnosus ◽

Type I ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Α Helix

Abstract Lpt is a 29 amino acid long type I toxin identified in the plasmid DNA of wild Lactobacillus rhamnosus strains isolated from food. We previously reported that transcription of the encoding gene was upregulated under nutritional starvation conditions mimicking cheese ripening environment. The heterologous expression of the Lpt peptide in E. coli resulted in cell growth inhibition, nucleoid condensation and compromised integrity of the cell membrane. Fusion of the Lpt peptide with the fluorescent protein mCherry allowed to visualize the accumulation of the peptide into the membrane, while mutagenesis experiments showed that either the insertion of a negatively charged amino acid into the hydrophobic α-helix or deletion of the hydrophilic C-terminal region, leads to a non-toxic peptide. AFM imaging of Lpt expressing E. coli cells has revealed the presence of surface defects that are compatible with the loss of portions of the outer membrane bilayer. This observation provides support for the so-called “carpet” model, by which the Lpt peptide is supposed to destabilize the phospholipid packing through a detergent-like mechanism leading to the removal of small patches of bilayer through micellization.

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Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina

Biochemical Journal ◽

10.1042/bj3380211 ◽

1999 ◽

Vol 338 (1) ◽

pp. 211-219 ◽

Cited By ~ 8

Author(s):

Lu-Ping CHOW ◽

Ming-Huei CHOU ◽

Cheng-Ying HO ◽

Chyh-Chong CHUANG ◽

Fu-Ming PAN ◽

...

Keyword(s):

Amino Acid ◽

Tertiary Structure ◽

Biological Effects ◽

Homology Modelling ◽

Peptide Sequence ◽

Type I ◽

Ribosome Inactivating Protein ◽

Signal Peptide Sequence ◽

Apparent Homogeneity ◽

A Chain

The seeds of the plant Trichosanthes anguina contain a type I ribosome-inactivating protein (RIP), designated trichoanguin, which was purified to apparent homogeneity by the combined use of ion-exchange chromatographies, i.e. first with DE-52 cellulose and then with CM-52 cellulose. The protein was found to be a glycoprotein with a molecular mass of 35 kDa and a pI of 9.1. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC50 of 0.08 nM, but only weakly that of HeLa cells, with an IC50 of 6 µM. Trichoanguin cleaves at the A4324 site of rat 28 S rRNA by its N-glycosidase activity. The cDNA of trichoanguin consists of 1039 nt and encodes an open reading frame coding for a polypeptide of 294 amino acid residues. The first 19 residues of this polypeptide encode a signal peptide sequence and the last 30 residues comprise an extension at its C-terminus. There are four potential glycosylation sites, located at Asn-51, Asn-65, Asn-201 and Asn-226. A comparison of the amino acid sequence of trichoanguin with those of RIPs such as trichosanthin, α-momorcharin, ricin A-chain and abrin A-chain reveals 55%, 48%, 36% and 34% identity respectively. Molecular homology modelling of trichoanguin indicates that its tertiary structure closely resembles those of trichosanthin and α-momorcharin. The large structural similarities might account for their common biological effects such as an abortifacient, an anti-tumour agent and anti-HIV-1 activities. Trichoanguin contains two cysteine residues, Cys-32 and Cys-155, with the former being likely to be located on the protein surface, which is directly amenable for conjugation with antibodies to form immunoconjugates. It is therefore conceivable that trichoanguin might be a better type I RIP than any other so far examined for the preparation of immunotoxins, with a great potential for application as an effective chemotherapeutic agent for the treatment of cancer.

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Molecular cloning and functional characterization of a type-I neurotensin receptor (NTR) and a novel NTR from the bullfrog brain

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01709 ◽

2005 ◽

Vol 34 (3) ◽

pp. 793-807 ◽

Cited By ~ 11

Author(s):

J H Li ◽

F Sicard ◽

M A Salam ◽

M Baek ◽

J LePrince ◽

...

Keyword(s):

Amino Acid ◽

Mammalian Species ◽

Functional Characterization ◽

High Sensitivity ◽

Structural Features ◽

Amino Acid Identity ◽

Type I ◽

Pharmacological Properties ◽

Vertebrate Lineage

Neurotensin (NT) is a tridecapeptide that functions as a neurotransmitter and neuromodulator in the nervous system. To date, three different types of NT receptor (NTR), NTR1, NTR2 and NTR3, have been identified only in mammalian species. In the present study we isolated the cDNAs for an NTR1 and a novel NTR in the bullfrog brain, designated bfNTR1 and bfNTR4 respectively. bfNTR1 and bfNTR4 encode 422- and 399-amino acid residue proteins respectively. bfNTR1 has a 64% amino acid identity with mammalian NTR1, and 34–37% identity with mammalian NTR2. bfNTR4 exhibits 43% and 45–47% identity with mammalian NTR1 and NTR2 respectively. Both receptors are mainly expressed in the brain and pituitary. bfNTR1 triggers both CRE-luc, a protein kinase A (PKA)-specific reporter, and c-fos-luc, a PKC-specific reporter, activities, indicating that bfNTR1 can activate PKA- and PKC-linked signaling pathways. However, bfNTR4 appears to be preferentially coupled to the PKA-linked pathway as it induces a higher CRE-luc activity than c-fos-luc activity. bfNTRs exhibit different pharmacological properties as compared with mammalian NTRs. Mammalian NTR1 but not NTR2 responds to NT, whereas both bfNTR1 and bfNTR4 show a high sensitivity to NT. SR 48692 and SR 142948A, antagonists for mammalian NTR1 but agonists for mammalian NTR2, function as antagonists for both bfNTR1 and bfNTR4. In conclusion, this report provides the first molecular, pharmacological and functional characterization of two NTRs in a non-mammalian vertebrate. These data should help to elucidate the phylogenetic history of the G protein-coupled NTRs in the vertebrate lineage as well as the structural features that determine their pharmacological properties.

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Type I Ribosome-Inactivating Proteins from Saponaria officinalis

Toxic Plant Proteins - Plant Cell Monographs ◽

10.1007/978-3-642-12176-0_4 ◽

2010 ◽

pp. 55-78 ◽

Cited By ~ 7

Author(s):

Alessio Lombardi ◽

Richard S. Marshall ◽

Carmelinda Savino ◽

Maria Serena Fabbrini ◽

Aldo Ceriotti

Keyword(s):

Type I ◽

Ribosome Inactivating Proteins ◽

Saponaria Officinalis

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An unusual type I ribosome-inactivating protein from Agrostemma githago L.

Scientific Reports ◽

10.1038/s41598-020-72282-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Christoph Weise ◽

Achim Schrot ◽

Leonie T. D. Wuerger ◽

Jacob Adolf ◽

Roger Gilabert-Oriol ◽

...

Keyword(s):

Current Knowledge ◽

Ricinus Communis ◽

Herbaceous Plant ◽

Type I ◽

Ribosome Inactivating Protein ◽

Ribosome Inactivating Proteins ◽

E Coli ◽

The Past ◽

First Time ◽

Unusual Type

Abstract Agrostemma githago L. (corn cockle) is an herbaceous plant mainly growing in Europe. The seeds of the corn cockle are toxic and poisonings were widespread in the past by consuming contaminated flour. The toxic principle of Agrostemma seeds was attributed to triterpenoid secondary metabolites. Indeed, this is in part true. However Agrostemma githago L. is also a producer of ribosome-inactivating proteins (RIPs). RIPs are N-glycosylases that inactivate the ribosomal RNA, a process leading to an irreversible inhibition of protein synthesis and subsequent cell death. A widely known RIP is ricin from Ricinus communis L., which was used as a bioweapon in the past. In this study we isolated agrostin, a 27 kDa RIP from the seeds of Agrostemma githago L., and determined its full sequence. The toxicity of native agrostin was investigated by impedance-based live cell imaging. By RNAseq we identified 7 additional RIPs (agrostins) in the transcriptome of the corn cockle. Agrostin was recombinantly expressed in E. coli and characterized by MALDI-TOF–MS and adenine releasing assay. This study provides for the first time a comprehensive analysis of ribosome-inactivating proteins in the corn cockle and complements the current knowledge about the toxic principles of the plant.

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The design and catalytic performance of molybdenum active sites on an MCM-41 framework for the aerobic oxidation of 5-hydroxymethylfurfural to 2,5-diformylfuran

Catalysis Science & Technology ◽

10.1039/c8cy02291g ◽

2019 ◽

Vol 9 (3) ◽

pp. 811-821 ◽

Cited By ~ 7

Author(s):

Zhao-Meng Wang ◽

Li-Juan Liu ◽

Bo Xiang ◽

Yue Wang ◽

Ya-Jing Lyu ◽

...

Keyword(s):

Catalytic Activity ◽

Active Sites ◽

Aerobic Oxidation ◽

Catalytic Performance ◽

Mcm 41

The catalytic activity decreases as –(SiO)3Mo(OH)(O) > –(SiO)2Mo(O)2 > –(O)4–MoO.

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Polymer structure and catalytic activity of ion exchangers

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19811577 ◽

1981 ◽

Vol 46 (7) ◽

pp. 1577-1587 ◽

Cited By ~ 8

Author(s):

Karel Jeřábek

Keyword(s):

Catalytic Activity ◽

Ethyl Acetate ◽

Active Sites ◽

Crosslinking Agent ◽

Reaction Medium ◽

Polymer Structure ◽

Local Concentration ◽

Ion Exchangers ◽

Styrene Divinylbenzene ◽

Kinetics Of

Catalytic activity of ion exchangers prepared by partial sulphonation of styrene-divinylbenzene copolymers in reesterifications of ethyl acetate by methanol and propanol, hydrolysis of ethyl acetate and in synthesis of bisphenol A has been compared with data on polymer structure of these catalysts and with distribution of the crosslinking agent, divinylbenzene, calculated from literature data on kinetics of copolymerisation of styrene with divinylbenzene. It was found that the polymer structure of ion exchangers influences catalytic activity predominantly by changing the local concentration of acid active sites. The results obtained indicated that the effect of transport phenomena on the rate of catalytic reactions does not depend on the degree of swelling of the ion exchangers in reaction medium but it is mainly dependent on the relative affinity of reaction components to the acid groups or to the polymer skeleton.

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Oscillatory Behaviour of Ni Supported on ZrO2 in the Catalytic Partial Oxidation of Methane as Determined by Activation Procedure

Materials ◽

10.3390/ma14102495 ◽

2021 ◽

Vol 14 (10) ◽

pp. 2495

Author(s):

Daniela Pietrogiacomi ◽

Maria Cristina Campa ◽

Ida Pettiti ◽

Simonetta Tuti ◽

Giulia Luccisano ◽

...

Keyword(s):

Catalytic Activity ◽

Partial Oxidation ◽

Active Sites ◽

Direct Reduction ◽

Partial Oxidation Of Methane ◽

Catalytic Partial Oxidation ◽

Oxidation Of Methane ◽

Short Contact Time ◽

Oxidation Reduction ◽

Ni Species

Ni/ZrO2 catalysts, active and selective for the catalytic partial oxidation of methane to syngas (CH4-CPO), were prepared by the dry impregnation of zirconium oxyhydroxide (Zhy) or monoclinic ZrO2 (Zm), calcination at 1173 K and activation by different procedures: oxidation-reduction (ox-red) or direct reduction (red). The characterization included XRD, FESEM, in situ FTIR and Raman spectroscopies, TPR, and specific surface area measurements. Catalytic activity experiments were carried out in a flow apparatus with a mixture of CH4:O2 = 2:1 in a short contact time. Compared to Zm, Zhy favoured the formation of smaller NiO particles, implying a higher number of Ni sites strongly interacting with the support. In all the activated Ni/ZrO2 catalysts, the Ni–ZrO2 interaction was strong enough to limit Ni aggregation during the catalytic runs. The catalytic activity depended on the activation procedures; the ox-red treatment yielded very active and stable catalysts, whereas the red treatment yielded catalysts with oscillating activity, ascribed to the formation of Niδ+ carbide-like species. The results suggested that Ni dispersion was not the main factor affecting the activity, and that active sites for CH4-CPO could be Ni species at the boundary of the metal particles in a specific configuration and nuclearity.

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