scholarly journals Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni

2018 ◽  
Vol 365 (16) ◽  
Author(s):  
Breeanna Mintmier ◽  
Jennifer M McGarry ◽  
Courtney E Sparacino-Watkins ◽  
Joseph Sallmen ◽  
Katrin Fischer-Schrader ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Campylobacter Jejuni ◽  
Biochemical Characterization ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Iron Sulfur Cluster ◽  
Periplasmic Nitrate Reductase ◽  
Isolation And Characterization ◽  
Gastrointestinal Pathogen

AbstractCampylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s−1 and a KM (nitrate) of 3.40 ± 0.44 μM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

scholarly journals Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

2012 ◽  
Vol 86 (16) ◽  
pp. 8375-8387 ◽  
Author(s):  
Marie Galloux ◽  
Bogdan Tarus ◽  
Ilfad Blazevic ◽  
Jenna Fix ◽  
Stéphane Duquerroy ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Interaction ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Interaction Domain ◽  
Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris

2004 ◽  
Vol 37 (1) ◽  
pp. 61-71 ◽  
Author(s):  
Guillaume G Barbier ◽  
Rama C Joshi ◽  
Ellen R Campbell ◽  
Wilbur H (Bill) Campbell
Keyword(s):  
Pichia Pastoris ◽  
Nitrate Reductase ◽  
Biochemical Characterization

Isolation and characterization of nitrate reductase-deficient mutants in tomato (Lycopersicon esculentum Mill.)

1991 ◽  
Vol 227 (3) ◽  
pp. 458-464 ◽  
Author(s):  
Herman C. H. Schoenmakers ◽  
Maarten Koornneef ◽  
Sjefke J. H. M. Alefs ◽  
Wendy F. M. Gerrits ◽  
Dianne van der Kop ◽  
...  
Keyword(s):  
Lycopersicon Esculentum ◽  
Nitrate Reductase ◽  
Lycopersicon Esculentum Mill ◽  
Isolation And Characterization

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

1998 ◽  
Vol 140 (2) ◽  
pp. 311-318 ◽  
Author(s):  
ROLAND MARMEISSE ◽  
PATRICIA JARGEAT ◽  
FRANCOISE WAGNER ◽  
GILLES GAY ◽  
JEAN-CLAUDE DEBAUD
Keyword(s):  
Nitrate Reductase ◽  
Ectomycorrhizal Fungus ◽  
Hebeloma Cylindrosporum ◽  
Isolation And Characterization

Biochemical characterization of Campylobacter jejuni PNPase, an exoribonuclease important for bacterial pathogenicity

Biochimie ◽  
2018 ◽  
Vol 147 ◽  
pp. 70-79 ◽  
Author(s):  
Jorge Casinhas ◽  
Rute G. Matos ◽  
Nabila Haddad ◽  
Cecília M. Arraiano
Keyword(s):  
Campylobacter Jejuni ◽  
Biochemical Characterization ◽  
Bacterial Pathogenicity

scholarly journals Biochemical characterization and essentiality ofPlasmodiumfumarate hydratase

2017 ◽  
Author(s):  
Vijay Jayaraman ◽  
Arpitha Suryavanshi ◽  
Pavithra Kalale ◽  
Jyothirmai Kunala ◽  
Hemalatha Balaram
Keyword(s):  
Biochemical Characterization ◽  
Tricarboxylic Acid Cycle ◽  
Fumarate Hydratase ◽  
Class I ◽  
Iron Sulfur Cluster ◽  
Mercaptosuccinic Acid ◽  
Nanomolar Range ◽  
Strain Dependent ◽  
Selection Of

ABSTRACTPlasmodium falciparum(Pf), the causative agent of malaria has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a well-known reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme. Fumarate, generated in large quantities in the parasite as a byproduct of AMP synthesis is converted to malate by the action of FH, and subsequently used in the generation of the key metabolites oxaloacetate, aspartate and pyruvate. Here we report on the kinetic characterization of purified recombinant PfFH, functional complementation offhdeficiency inEscherichia coliand mitochondrial localization in the parasite. The substrate analog, mercaptosuccinic acid was found to be a potent inhibitor of PfFH with a Kivalue in the nanomolar range. Knockout of thefhgene was not possible inP. bergheiwhen drug-selection of the transfectants was performed in BALB/c mice while the gene was amenable to knockout when C57BL/6 mice were used as host, thereby indicating mouse-strain dependent essentiality of thefhgene to the parasite.

scholarly journals Biochemical Characterization of Laboratory Mutants of Extended-Spectrum β-Lactamase TEM-60

2004 ◽  
Vol 48 (9) ◽  
pp. 3579-3582 ◽  
Author(s):  
Bibiana Caporale ◽  
Nicola Franceschini ◽  
Mariagrazia Perilli ◽  
Bernardetta Segatore ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Extended Spectrum

ABSTRACT Three mutants of the extended-spectrum β-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

scholarly journals Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha

1994 ◽  
Vol 220 (1) ◽  
pp. 117-124 ◽  
Author(s):  
Ben C. BERKS ◽  
David J. RICHARDSON ◽  
Carol ROBINSON ◽  
Ann REILLY ◽  
Robin T. APLIN ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Purification And Characterization ◽  
Periplasmic Nitrate Reductase ◽  
Thiosphaera Pantotropha
Sign in / Sign up

Export Citation Format

Share Document