Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase

Protein Expression and Purification
R L Koder, Anne-Frances Miller

Abstract

Bacterial nitroreductases have generated much interest recently due to their central roles in both nitroaromatic bioremediation and nitroaromatic toxicity, mutagenicity, and carcinogenicity. Enterobacter cloacae nitroreductase (NR) has been subcloned into the pET overexpression system and purified to homogeneity via a four-step procedure resulting in a final yield of 65.7 mg per liter. Overexpression in minimal media containing 15NH4Cl as the sole source of nitrogen yielded 37.6 mg per liter of homogenous NR containing > 99 atom % 15N. A series of melting curves generated under a variety of solvent conditions established the optimal conditions for NR stability as pH 7.5, low ionic strength phosphate buffer. A two-dimensional 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance spectrum demonstrates this enzyme to be amenable to study by high-resolution multidimensional NMR in combination with amino-acid-specific isotopic labeling. Optical spectra of the purified enzyme suggest that the noncovalently bound flavin mononucleotide cofactor binds in a hydrophobic environment and is in the neutral and anionic protonation states in the oxidized and two-electron reduced oxidation states, respectively. NR exhibits a ...Continue Reading

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Nov 1, 1979·European Journal of Biochemistry·A J VisserC Veeger
Jan 1, 1976·Analytical Biochemistry·A Bensadoun, D Weinstein
Aug 1, 1992·Journal of General Microbiology·P E GroenewegenJ A de Bont
Jun 1, 1990·Microbiological Reviews·B J Bachmann
Sep 1, 1974·Journal of Bacteriology·F C NeidhardtD F Smith
Jan 1, 1969·Photochemistry and Photobiology·J Koziol
Aug 25, 1995·Biochemical Pharmacology·G M AnlezarkA Williams
Aug 1, 1993·Applied and Environmental Microbiology·S F Nishino, J C Spain
Apr 1, 1993·Protein Expression and Purification·J T MooreG F Maley
Nov 1, 1995·Journal of Biomolecular NMR·F DelaglioA Bax
Jan 1, 1995·Annual Review of Microbiology·J C Spain
Jan 1, 1996·Environmental and Molecular Mutagenesis·K T ChungM W Chou
Dec 1, 1995·European Journal of Cancer : Official Journal for European Organization for Research and Treatment of Cancer (EORTC) [and] European Association for Cancer Research (EACR)·J A BridgewaterM K Collins

❮ Previous
Next ❯

Citations

Nov 7, 2003·Applied and Environmental Microbiology·Venkateswarlu KadiyalaJim C Spain
Mar 22, 2008·FEMS Microbiology Reviews·María Dolores RoldánConrado Moreno-Vivián
Jan 25, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Anne-Frances MillerAndreas S Bommarius
Feb 24, 2015·Chembiochem : a European Journal of Chemical Biology·Jonathan T ParkAndreas S Bommarius
Apr 8, 2014·The Journal of Biological Chemistry·Warintra PitsawongAnne-Frances Miller
Jan 24, 2002·The Journal of Biological Chemistry·Chad A HaynesDavid W Rodgers
Jul 26, 2002·Archives of Biochemistry and Biophysics·Henrikas NivinskasNarimantas Cenas
Nov 27, 2002·Biochemistry·Ronald L KoderAnne-Frances Miller

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.