Molecular basis of enzymatic nitrogen-nitrogen formation by a family of zinc-binding cupin enzymes

AbstractMolecules with a nitrogen-nitrogen (N-N) bond in their structures exhibit various biological activities and other unique properties. A few microbial proteins are recently emerging as dedicated N-N bond forming enzymes in natural product biosynthesis. However, the details of these biochemical processes remain largely unknown. Here, through in vitro biochemical characterization and computational studies, we report the molecular basis of hydrazine bond formation by a family of di-domain enzymes. These enzymes are widespread in bacteria and sometimes naturally exist as two standalone enzymes. We reveal that the methionyl-tRNA synthase-like domain/protein catalyzes ATP-dependent condensation of two amino acids substrates to form a highly unstable ester intermediate, which is subsequently captured by the zinc-binding cupin domain/protein and undergoes redox-neutral intramolecular rearrangement to give the N-N bond containing product. These results provide important mechanistic insights into enzymatic N-N bond formation and should facilitate future development of novel N-N forming biocatalyst.

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The Stemness of Human Ovarian Granulosa Cells and the Role of Resveratrol in the Differentiation of MSCs—A Review Based on Cellular and Molecular Knowledge

Cells ◽

10.3390/cells9061418 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1418 ◽

Cited By ~ 1

Author(s):

Malgorzata Jozkowiak ◽

Greg Hutchings ◽

Maurycy Jankowski ◽

Katarzyna Kulcenty ◽

Paul Mozdziak ◽

...

Keyword(s):

Signaling Pathways ◽

Granulosa Cells ◽

Current Knowledge ◽

Biological Activities ◽

Primordial Follicle ◽

Biochemical Processes ◽

Ovarian Granulosa Cells ◽

Physiological Properties

Ovarian Granulosa Cells (GCs) are known to proliferate in the developing follicle and undergo several biochemical processes during folliculogenesis. They represent a multipotent cell population that has been differentiated to neuronal cells, chondrocytes, and osteoblasts in vitro. However, progression and maturation of GCs are accompanied by a reduction in their stemness. In the developing follicle, GCs communicate with the oocyte bidirectionally via gap junctions. Together with neighboring theca cells, they play a crucial role in steroidogenesis, particularly the production of estradiol, as well as progesterone following luteinization. Many signaling pathways are known to be important throughout the follicle development, leading either towards luteinization and release of the oocyte, or follicular atresia and apoptosis. These signaling pathways include cAMP, PI3K, SMAD, Hedgehog (HH), Hippo and Notch, which act together in a complex manner to control the maturation of GCs through regulation of key genes, from the primordial follicle to the luteal phase. Small molecules such as resveratrol, a phytoalexin found in grapes, peanuts and other dietary constituents, may be able to activate/inhibit these signaling pathways and thereby control physiological properties of GCs. This article reviews the current knowledge about granulosa stem cells, the signaling pathways driving their development and maturation, as well as biological activities of resveratrol and its properties as a pro-differentiation agent.

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Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper

Biochemical Journal ◽

10.1042/bj3260853 ◽

1997 ◽

Vol 326 (3) ◽

pp. 853-859 ◽

Cited By ~ 46

Author(s):

Sergio LIZANO ◽

Bruno LOMONTE ◽

Jay W. FOX ◽

José Maréa GUTIÉRREZ

Keyword(s):

Phospholipase A2 ◽

Molecular Mass ◽

Biochemical Characterization ◽

Biological Activities ◽

Sequence Similarity ◽

Gel Filtration ◽

Cytolytic Activity ◽

Bothrops Asper

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23–25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23–25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I–IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

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Ultrasound Assisted Synthesis of 3-(het)aryl Isocoumarin Derivatives and their in vitro Pharmacological Evaluation

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557519666190130163708 ◽

2019 ◽

Vol 19 (10) ◽

pp. 842-850 ◽

Cited By ~ 1

Author(s):

Gutta Lakshmi Prasanna ◽

V.D. Nagendra Kumar Abbaraju ◽

Yarlagadda Bharath ◽

Mandava V. Basaveswara Rao ◽

Manojit Pal

Keyword(s):

Boronic Acid ◽

Biological Activities ◽

Ultrasound Irradiation ◽

Bond Forming ◽

Pharmacological Evaluation ◽

Tnf Α ◽

Ic50 Value ◽

Ultrasound Assisted ◽

Boronic Acid Derivatives

Background: In view of numerous biological activities of 3-substituted isocoumarins a number of analogues based on this scaffold were synthesized for their in vitro pharmacological evaluation. Methods: The syntheses of 3-substituted isocoumarins were carried out via a Pd/C-catalyzed Suzuki- Miyaura coupling of 3-chloroisochromen-1-one with a range of boronic acid derivatives. This C-C bond forming reaction was facilitated by ultrasound irradiation to afford the desired products in good yields. A number of 3-(het)aryl isocoumarin derivatives were prepared by using this methodology and subsequently tested for their TNF-α inhibitory properties in vitro followed by cytotoxicities via the MTT assay. Results: Several compounds showed inhibition of TNF-α with one compound showing an IC50 value of 9.01±1.25 µM. Three compounds also showed promising cytotoxic properties against two cancer cell lines with IC50 ~ 0.9-2.7 µM. Conclusion: The isocoumarin framework could be an effective template for the design and discovery of new inhibitor of TNF-α for the potential treatment of related diseases.

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In VitroStudies on Thioether Bond Formation betweenHydrogenobacter thermophilusApocytochromec552with Metalloprotoporphyrin Derivatives

Journal of Biological Chemistry ◽

10.1074/jbc.m408637200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45347-45353 ◽

Cited By ~ 19

Author(s):

Oliver Daltrop ◽

Stuart J. Ferguson

Keyword(s):

Metal Ion ◽

De Novo ◽

Bond Formation ◽

Protoporphyrin Ix ◽

Prosthetic Group ◽

Forming Mechanism ◽

Bond Forming ◽

Potential Applications ◽

Cysteine Thiols

Previously,in vitroformation of thioether bonds betweenHydrogenobacter thermophilusapocytochromec552and Fe-protoporphyrin IX has been demonstrated (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002)Proc. Natl. Acad. Sci. U. S. A.99, 7872–7876). Now we report studies on the reaction between the metalloderivatives Zn-, Co-, and Mn-protoporphyrin IX and the cysteine thiols ofH. thermophilusapocytochromec552. All of these metalloporphyrins were capable of forminga“b-type cytochrome” state in which the hydrophobic prosthetic group is bound non-covalently. Zn(II)-protoporphyrin IX attached to the polypeptide covalently in the presence of either dithiothreitol or tri(2-carboxyethyl)phosphine to keep the thiol moieties reduced. These data show that the chemical nature of the thiol-reducing agent does not interfere with the thioether bond-forming mechanism. Mn-porphyrin could only react with the protein in the divalent state of the metal ion. Co-porphyrin did not react with the cysteine thiols of the apocytochrome in either oxidation state of the metal. In the absence of a metal (i.e.protoporphyrin IX itself), no reactivity toward apocytochrome is observed. These results have significant implications for the chemical requirements for thioether bond formation of heme vinyl groups to cysteine thiols and also have potential applications inde novodesign of metalloproteins.

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Biochemical characterization and in vitro biological activities of the epithelial cell extracts from Hypanus dipterurus spine

Toxicon ◽

10.1016/j.toxicon.2020.08.032 ◽

2020 ◽

Vol 187 ◽

pp. 129-135

Author(s):

Estrella Marrón-Grijalba ◽

César Salvador Cardona-Félix ◽

Víctor Hugo Cruz-Escalona ◽

Mauricio Muñoz-Ochoa ◽

Claudio Cabral-Romero ◽

...

Keyword(s):

Epithelial Cell ◽

Biochemical Characterization ◽

Biological Activities ◽

Cell Extracts

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The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146217 ◽

1993 ◽

Vol 51 ◽

pp. 74-75

Author(s):

Jason R. Swedlow ◽

Neil Osheroff ◽

Tim Karr ◽

John W. Sedat ◽

David A. Agard

Keyword(s):

Topoisomerase Ii ◽

Biochemical Characterization ◽

Developmental Cycle ◽

Dna Topoisomerase Ii ◽

Chromosomal Distribution ◽

Dna Topoisomerase ◽

Ideal System ◽

Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

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Dwindling status of Epimedium Elatum (Morren & Decne) and its geographical distribution in Kashmir Himalaya, India

Current Botany ◽

10.25081/cb.2018.v9.20180106 ◽

2018 ◽

pp. 47-52

Keyword(s):

Medicinal Plant ◽

Biological Activities ◽

Conservation Status ◽

Natural Populations ◽

Culture Techniques ◽

Kashmir Himalayas ◽

Near Future ◽

First Time ◽

Tissue Culture Techniques

Epimedium elatum (Morren & Decne) of family Berberidaceace is a rare perennial medicinal plant, endemic to high altitude forests of Northwestern Himalayas in India. Ethnobotanically, it has been used as an ingredient for treatment of bone-joint disorders, impotence and kidney disorders in Kashmir Himalayas. Phytochemically, it is rich in Epimedin ABC and Icariin; all of these have been demonstrated to possess remarkable biological activities like PDE-5 inhibition (treatment of erectile dysfunction), anticancer, antiosteoporosis antioxidant and antiviral properties. The present investigation reports its traditional usage, comprehensive distribution and conservation status from twenty ecogeographical regions in Kashmir Himalayas, India. The species was reported from Gurez valley for the first time. Numerous threats like excessive grazing, deforestration, habitat fragmentation, tourism encroachment, landslides and excessive exploitation have decreased its natural populations in most of the surveyed habitats. Consequently, its existence may become threatened in near future if timely conservation steps are not taken immediately by concerned stakeholders involved in medicinal plant research. Moreover, use of plant tissue culture techniques is recommended for development of its in vitro propagation protocols. Therefore, introduction of this medicinal plant in botanical gardens, protected sites and development of monitoring programmes are needed for its immediate conservation in Northwestern Himalayas, India.

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Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

10.26434/chemrxiv.12710159.v1 ◽

2020 ◽

Author(s):

Sean A. Newmister ◽

Kinshuk Raj Srivastava ◽

Rosa V. Espinoza ◽

Kersti Caddell Haatveit ◽

Yogan Khatri ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Molecular Basis ◽

Hydrophobic Interactions ◽

Cytochromes P450 ◽

Targeted Mutagenesis ◽

P450 Monooxygenase ◽

Bond Functionalization ◽

Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.

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Radical Capture at Ni(II) Complexes: C-C, C-N, and C-O Bond Formation

10.26434/chemrxiv.8115860.v1 ◽

2019 ◽

Author(s):

Abolghasem (Gus) Bakhoda ◽

Stefan Wiese ◽

Christine Greene ◽

Bryan C. Figula ◽

Jeffery A. Bertke ◽

...

Keyword(s):

Functional Groups ◽

Bond Formation ◽

Dft Studies ◽

Bond Forming ◽

Aryl Amines ◽

Shed Light ◽

Competing Pathways

The dinuclear b-diketiminato NiIItert-butoxide {[Me3NN]Ni}2(μ-OtBu)2 (2), synthesized from [Me3NN]Ni(2,4-lutidine) (1) and di-tert-butylperoxide, is a versatile precursor for the synthesis of a series of NiIIcomplexes [Me3NN]Ni-FG to illustrate C-C, C-N, and C-O bond formation at NiII via radicals. {[Me3NN]Ni}2(μ-OtBu)2 reacts with nitromethane, alkyl and aryl amines, acetophenone, benzamide, ammonia and phenols to deliver corresponding mono- or dinuclear [Me3NN]Ni-FG species (FG = O2NCH2, R-NH, ArNH, PhC(O)NH, PhC(O)CH2, NH2and OAr). Many of these NiII complexes are capable of capturing the benzylic radical PhCH(•)CH3 to deliver corresponding PhCH(FG)CH3 products featuring C-C, C-N or C-O bonds. DFT studies shed light on the mechanism of these transformations and suggest two competing pathways that depend on the nature of the functional groups. These radical capture reactions at [NiII]-FG complexes outline key C-C, C-N, and C-O bond forming steps and suggest new families of nickel radical relay catalysts.

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Evaluation of the In Vivo Acute Toxicity and In Vitro Hemolytic and Immunomodulatory Activities of the Moringa oleifera Flower Trypsin Inhibitor (MoFTI)

Protein and Peptide Letters ◽

10.2174/0929866527999201113105858 ◽

2020 ◽

Vol 27 ◽

Author(s):

Leydianne Leite de Siqueira Patriota ◽

Dayane Kelly Dias do Nascimento Santos ◽

Bárbara Rafaela da Silva Barros ◽

Lethícia Maria de Souza Aguiar ◽

Yasmym Araújo Silva ◽

...

Keyword(s):

Acute Toxicity ◽

Trypsin Inhibitor ◽

Hemolytic Activity ◽

Moringa Oleifera ◽

Biological Activities ◽

Hematological Parameters ◽

Behavioral Alterations ◽

Female Mice

Background: Protease inhibitors have been isolated from plants and present several biological activities, including immunomod-ulatory action. Objective: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes. Methods: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15–240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO). Results: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15–30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as ΔΨm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release. Conclusion: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.

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