scholarly journals Lopap, a prothrombin activator from Lonomia obliqua belonging to the lipocalin family: recombinant production, biochemical characterization and structure–function insights

2006 ◽  
Vol 398 (2) ◽  
pp. 295-302 ◽  
Author(s):  
Cleyson Valença Reis ◽  
Sonia Aparecida Andrade ◽  
Oscar Henrique Pereira Ramos ◽  
Celso Raul Romero Ramos ◽  
Paulo Lee Ho ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Serine Protease ◽  
Calcium Binding ◽  
Biochemical Characterization ◽  
Structural Features ◽  
Reading Frame ◽  
Molecular Features ◽  
Recombinant Production ◽  
Lonomia Obliqua

Using a cDNA library made from Lonomia obliqua caterpillar bristles, we identified a transcript with a 603 bp open reading frame. The deduced protein corresponds to Lopap, a prothrombin activator previously isolated by our group from the bristles of this species. The mature protein is composed by 185 amino acids and shares similarity with members of the lipocalin family. The cDNA encoding the mature form was amplified by PCR, subcloned into pAE vector and used to transform Escherichia coli BL21(DE3) cells. As for the native Lopap, the recombinant fusion protein shows enzymatic activity, promotes prothrombin hydrolysis, generates fragments similar to prethrombin-2 and fragment 1.2 as intermediates, and generates thrombin as the final product. In addition, structural bioinformatics studies indicated several interesting molecular features, including the residues that could be responsible for Lopap's serine protease-like activity and the role of calcium binding in this context. Such catalytic activity has never been found in other members of the lipocalin family. This is the first report describing the recombinant production and biochemical characterization of a Lonomia obliqua lipocalin, as well as the structural features that could be responsible for its serine protease-like catalytic activity.

scholarly journals Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

2019 ◽  
Author(s):  
Murtala Muhammad ◽  
Yangyang Li ◽  
Siyu Gong ◽  
Yanmin Shi ◽  
Jiansong Ju ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Antibiotic Activity ◽  
Alanine Racemase ◽  
Streptococcus Iniae ◽  
Gram Negative Bacteria ◽  
Reading Frame ◽  
New Antibiotics ◽  
Serious Disease ◽  
Zoonotic Bacteria

ABSTRACTStreptococcus iniaeis a pathogenic and zoonotic bacteria that impacted high mortality to many fish species, as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5′-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from the pathogenic strain ofS. iniae, determined its biochemical characteristics and inhibitors. Thealrgene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The optimal enzyme activity occurred at 35°C and a pH of 9.5. The enzyme belongs to the PLP dependent enzymes family and is highly specific to L-alanine.S.iniaeAlr can be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parametersKmandVmaxof the enzyme were 33.11 mM, 2426 units/mg for L-alanine and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors, were determined and found to be effective against both gram positive and gram negative bacteria employed in this study. The important role of alanine racemase as a target of developing new antibiotics againstS. iniaehighlighted the usefulness of the enzyme for new antibiotics discovery.

scholarly journals Secondary structural features of the bacteriophage Mu-encoded A and B transposition proteins

1989 ◽  
Vol 263 (1) ◽  
pp. 19-23 ◽  
Author(s):  
G Chaconas ◽  
W D McCubbin ◽  
C M Kay
Keyword(s):  
Biochemical Characterization ◽  
Alpha Helix ◽  
Structural Features ◽  
Random Coil ◽  
Beta Sheet ◽  
Bacteriophage Mu ◽  
Intensive Study ◽  
Beta Turn ◽  
Biochemical Level

The role of the bacteriophage Mu-encoded A and B proteins is to direct the transposition of Mu DNA. These are the first active DNA transposition proteins to have been purified and their mechanism of action at the biochemical level is under intensive study. Structural studies on these proteins, however, have lagged behind their biochemical characterization. We report here near- and far-u.v. c.d. spectra for these proteins and their secondary structural features derived from these data. The Mu A protein appears to be composed of primarily beta-sheet (40%) with 24% alpha-helix, 9% beta-turn and 27% random coil. In contrast, the Mu B protein contains 55% alpha-helix with only 13% beta-sheet and 3+ beta-turn and 29% random coil. The near-u.v. c.d. spectrum of the A protein was not unusual; however, the profile of the B protein suggested either buried or restricted chromophores within the protein or short-range interactions between aromatic residues.

scholarly journals High-throughput mutagenesis reveals unique structural features of human ADAR1

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
SeHee Park ◽  
Erin E. Doherty ◽  
Yixuan Xie ◽  
Anil K. Padyana ◽  
Fang Fang ◽  
...  
Keyword(s):  
High Throughput ◽  
Rational Design ◽  
Biochemical Characterization ◽  
Structural Information ◽  
Structural Features ◽  
Adenosine Deaminases ◽  
Zinc Binding Site ◽  
Editing Activity ◽  
Deaminase Domain

Abstract Adenosine Deaminases that act on RNA (ADARs) are enzymes that catalyze adenosine to inosine conversion in dsRNA, a common form of RNA editing. Mutations in the human ADAR1 gene are known to cause disease and recent studies have identified ADAR1 as a potential therapeutic target for a subset of cancers. However, efforts to define the mechanistic effects for disease associated ADAR1 mutations and the rational design of ADAR1 inhibitors are limited by a lack of structural information. Here, we describe the combination of high throughput mutagenesis screening studies, biochemical characterization and Rosetta-based structure modeling to identify unique features of ADAR1. Importantly, these studies reveal a previously unknown zinc-binding site on the surface of the ADAR1 deaminase domain which is important for ADAR1 editing activity. Furthermore, we present structural models that explain known properties of this enzyme and make predictions about the role of specific residues in a surface loop unique to ADAR1.

Studies on role of calmodulin in Ca2+ regulation of rabbit ileal Na and Cl transport

1985 ◽  
Vol 248 (6) ◽  
pp. G726-G740 ◽  
Author(s):  
M. Donowitz ◽  
J. Wicks ◽  
J. L. Madara ◽  
G. W. Sharp
Keyword(s):  
Calcium Binding ◽  
Transport Process ◽  
Structural Features ◽  
Hydrophobic Properties ◽  
Calmodulin Antagonist ◽  
Camp Content ◽  
Cl Transport ◽  
Ileal Absorption ◽  
Stimulation Of

To study the role of calmodulin in regulation of rabbit ileal active electrolyte transport by Ca2+, the effects of the Ca2+-calmodulin antagonist naphthalenesulfonamides W12 and W13 and the weak Ca2+-calmodulin antagonist promethazine, a phenothiazine, were studied on basal ileal Na and Cl transport and on secretion stimulated by Ca2+ and by cAMP. The naphthalenesulfonamides and promethazine all stimulated basal ileal Na and Cl absorption. The stimulation of Na absorption was dependent on Cl in the bathing solutions, and the stimulation of Cl absorption was Na dependent. This suggests that the transport process stimulated was the neutral, linked NaCl absorptive process. W13, which is a better Ca2+-calmodulin antagonist but has similar hydrophobic properties to W12, stimulated active ileal absorption at a lower concentration than W12, suggesting that the naphthalenesulfonamide-induced stimulation of active ileal absorption was not due to the hydrophobic properties of these drugs but could be due to their effects on the calcium-binding protein calmodulin. W12, W13, and promethazine did not alter paracellular transport, not affecting dilution potentials or structural features of the paracellular pathway. W12 and W13 did not decrease the changes in active ileal Na and Cl transport caused by increasing ileal cAMP content or intracellular Ca2+. This suggests that calmodulin is not directly involved in the active electrolyte secretion caused by increased intestinal Ca2+ or cAMP.

scholarly journals The Potential Function of KIF17 in Large Yellow Croaker (Larimichthys crocea) Spermatid Remodeling: Molecular Characterization and Expression Pattern During Spermiogenesis

2021 ◽  
Author(s):  
Jingqian Wang ◽  
Zhao Liu ◽  
Xinming Gao ◽  
Chen Du ◽  
Congcong Hou ◽  
...  
Keyword(s):  
Stage Iv ◽  
Structural Similarity ◽  
Structural Features ◽  
Testicular Development ◽  
Large Yellow Croaker ◽  
Larimichthys Crocea ◽  
High Sequence Identity ◽  
Reading Frame ◽  
Perinuclear Cytoplasm

Abstract KIF17, which belongs to the kinesin-2 protein family, plays an indispensable role in mammalian spermiogenesis. However, the role of KIF17 in fish spermatid remodeling during spermiogenesis remains poorly understood. Therefore, we aimed to study the role of KIF17 in spermatid remodeling during Larimichthys crocea (L. crocea) spermiogenesis. The kif17 cDNA sequence, 3247 bp in length, was cloned from L. crocea testis, which consisted of a 347 bp 5ʹ-untranslated region (UTR), 413 bp 3ʹ -UTR, and 2487 bp open reading frame. Bioinformatic analyses revealed that KIF17 obtained from L. crocea (Lc-KIF17) exhibited a high sequence identity compared with those from other teleosts and possessed the structural features of other kinesin-2 proteins. Based on structural similarity, we speculate that the role of Lc-KIF17 may be similar to that of KIF17 in other animals. Lc-kif17 mRNA was diffusely expressed in L. crocea tissues and was highly expressed in the testis, especially at stage IV testicular development. Immunofluorescence analysis revealed that Lc-KIF17 signals colocalized with β-tubulin signals and migrated from the perinuclear cytoplasm to the side of the nucleus where the tail forms during spermiogenesis. These findings revealed that KIF17 may be involved in L. crocea spermiogenesis. In particular, KIF17 may participate in spermatid remodeling by interacting with perinuclear microtubules during L. crocea spermiogenesis. Collectively, this study contributes to an improved understanding of the mechanism underlying L. crocea spermiogenesis and provides a basis for further research on L. crocea reproduction and development.

Bimetallic Gold–Palladium vapour derived catalysts: The role of structural features on their catalytic activity

2012 ◽  
Vol 286 ◽  
pp. 224-236 ◽  
Author(s):  
Claudio Evangelisti ◽  
Eleonora Schiavi ◽  
Laura Antonella Aronica ◽  
Anna Maria Caporusso ◽  
Giovanni Vitulli ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Structural Features

Supported rhodium nanoparticles in catalysis: the role of stabilizers on catalytic activity and structural features

2003 ◽  
Vol 681 (1-2) ◽  
pp. 37-50 ◽  
Author(s):  
Giovanni Vitulli ◽  
Claudio Evangelisti ◽  
Paolo Pertici ◽  
Anna Maria Caporusso ◽  
Nicoletta Panziera ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Structural Features ◽  
Rhodium Nanoparticles

scholarly journals Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization

2010 ◽  
Vol 84 (21) ◽  
pp. 11310-11322 ◽  
Author(s):  
Kathryn M. Frietze ◽  
Samuel K. Campos ◽  
Adriana E. Kajon
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Structural Features ◽  
Open Reading Frames ◽  
Late Time ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Human Adenoviruses ◽  
Infected Cells

ABSTRACT Subspecies B1 human adenoviruses (HAdV-B1s) are important causative agents of acute respiratory disease, but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1s and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDα open reading frames (ORFs), and in the same location as the HAdV-C ADP/E3-11.6K ORF, HAdV-B1s carry ORFs E3-20.1K and E3-20.5K and a polymorphic third ORF, designated E3-10.9K, that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of the E3-10.9K ORF, we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed, C-terminally tagged (with enhanced green fluorescent protein [EGFP]) E3-10.9K and E3-9K localized primarily to the plasma membrane, while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N and O glycosylated. EGFP-tagged E3-4.8K, which lacked the hydrophobic domain, displayed diffuse cellular localization similar to that of the EGFP control. E3-10.9K transcripts from the major late promoter were detected at late time points postinfection. A C-terminally hemagglutinin-tagged version of E3-9K was detected by immunoprecipitation at late times postinfection in the membrane fraction of mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication, with potentially important functional implications for the documented ORF polymorphism.

Correction: Structural modeling and role of HAX-1 as a positive allosteric modulator of human serine protease HtrA2

2020 ◽  
Vol 477 (2) ◽  
pp. 459-459
Author(s):  
Lalith K. Chaganti ◽  
Shubhankar Dutta ◽  
Raja Reddy Kuppili ◽  
Mriganka Mandal ◽  
Kakoli Bose
Keyword(s):  
Serine Protease ◽  
Structural Modeling ◽  
Allosteric Modulator ◽  
Positive Allosteric Modulator
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