purification and properties of nitrite reductase from escherichia coli. by Kathleen Jane Coleman

Cover of: purification and properties of nitrite reductase from escherichia coli. | Kathleen Jane Coleman

Published by University of Birmingham in Birmingham .

Written in English

Read online

Edition Notes

Thesis (Ph.D.)- University of Birmingham, Dept of Biochemistry.

Book details

ID Numbers
Open LibraryOL20009300M

Download purification and properties of nitrite reductase from escherichia coli.

NADH-nitrite oxidoreductase (EC ) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis.

Yields of purified enzyme were low, mainly because Cited by:   Purification and properties of nitrite reductase from Escherichia coli K Coleman KJ, Cornish-Bowden A, Cole JA. NADH-nitrite oxidoreductase (EC ) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase by: Purification and properties of nitrite reductase from Escherichia coli K12 Article (PDF Available) in Biochemical Journal (2) December with 32 Reads How we measure 'reads'.

frombatch cultures ofEscherichia coli strain OR75Ch15,whichis partially constitutive for nitrite reductase synthesis. Yields ofpurified enzymewerelow, mainly because ofa large loss of activity during chromatography on DEAE-cellulose.

The quantitative separation ofcytochromec fromnitrite reductase activity resulted in anincrease inCited by: Purification and Properties of Nitrate Reductase from The membrane-bound enzyme nitrate reductase from Escherichia coli has been solubilized and purified fold.

The purified enzyme appeared homogeneous by polyacryl- nitrate to nitrite with methyl viologen as the donor. NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least fold and separated into two enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper.

Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems.

The two enzymes are encoded by distinct operons located within two different loci on theE. coli chromosome. The periplasmic nitrate reductase, NapA, from Escherichia coli was identified as a 90 kDa molybdoprotein which co-migrated during polyacrylamide gel electrophoresis with the di-haem c-type cytochrome, DNA sequence of the 5′ end of the napA gene and the N-terminal amino acid sequences of both NapA and NapB were determined.

The 36 residue leader peptide for NapA. Purification of the nitrate reductase. Enzyme purification was performed under aerobic conditions and at room temperature unless otherwise indicated.

Frozen cells were resuspended in 20 mM sodium phosphate buffer (pH ) and broken by sonication. The membrane fraction was collected by ultracentrifugation at× g for 60 min at 4°C.

reductase that we first studied was in bacteria belong- ing to the first group. Now with purpose of com- paring it with the nitrate reductase of facultative bacteria, we have purified the enzyme of E. coli. This paper describes the solubilization, purification and some physical and chemical properties of.

Coleman JK, Cornish-Bowden A, Cole JA () Purification and properties of nitrite reductase from Escherichia coli K Biochem J – Biochem J – Google Scholar. Kajie S, Anraku Y. Purification of a hexaheme cytochrome c from Escherichia coli K 12 and its properties as a nitrite reductase.

Eur J Biochem. Jan 15; (2)– Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

Nature. Aug 15; ()– Lanyi JK. D2. Coleman, A. Cornish-Bowden & J. Cole () Purification and properties of nitrite reductase from Escherichia coli K12 Biochem.– D3. Coleman, A. Cornish-Bowden & J. Cole () Activation of nitrite reductase from Escherichia coli K12 by oxidized nicotinamide–adenine dinucleotide Biochem.The nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed.

It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight Summary. Five mutants of Escherichia coli impaired on nitrite reduction were studied. All have lost NADH-nitrite reductase activity but have retained the capacity to synthesize all or part of their cytochrome c Three genes, nir C, nir D, and nir E were mapped at 26, and min, respectively.

Another gene, nir F was tentatively localized around 52 min. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two enzymes, periplasmic nitrate reductase (NapA) and periplasmic cytochrome c nitrite reductase (NrfA). The process is found in many enteric bacteria and may be important for anaerobic nitrate and nitrite respiration at low nitrate concentrations (1, 2).

Confirmatory assays for nitrite reductase showed that Frankia indeed reduced nitrite and used it as a nitrogen source. An Escherichia coli fosmid clone carrying the nirB region was able to grow better in the presence of 5 mM nitrite than without it, confirming the function of the genome region.

Nitrate and nitrite reduction are of paramount importance for nitrogen assimilation and anaerobic metabolism, and understanding the specific roles of each participating reductase is necessary to describe the biochemical balance that dictates cellular responses to their environments.

The soluble, cytoplasmic siroheme NADH-nitrite reductase (Nir) in Escherichia coli is necessary for nitrate. Sulphate-Reducing Bacteria - May nitrite reductase, catalyzed the reductions of both nitrite and hydroxylamine to ammonia, and the ratio of the two activities remained constant during partial purification (1).

Abstract. Escherichia coli can reduce nitrite to ammonium via a kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex. Recent structure-based spectropotentiometric studies are shedding light on the catalytic mechanism of NrfA; however, electron input into the enzyme has not been addressed biochemically.

The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure ( kDa/subunit) containing two copper atoms per monomer, one of.

Shin‐ichi KAJIE, Yasuhiro ANRAKU, Purification of a hexaheme cytochrome C from Escherichia coli K 12 and its properties as a nitrite reductase, European Journal of Biochemistry, /jtbx,2, (), (). Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons.

To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions.

NADH-nitrite oxidoreductase (EC ) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis.

Yields of purified enzyme were low, mainly because of a. A method was developed for urinary nitrate analysis utilizing an enzyme of Escherichia coli for the reduction of nitrate to nitrite.

The resulting nitrite was assayed by a standard diazotization procedure. Under the experimental conditions stoichiometric conversion of nitrate to nitrite was achieved.

@article{osti_, title = {Expression and purification of spinach nitrite reductase in E. coli}, author = {Bellissimo, D and Privalle, L}, abstractNote = {The study of structure-function relationships in nitrite reductase (NiR) by site-directed mutagenesis requires an expression system from which suitable quantities of active enzyme can be purified.

nitrite reductase, large subunit, NAD(P)H-binding, B, NirB, ECK, JW, b Product description nitrite reductase, large subunit; Component of dimer of large subunit of nitrite reductase; nitrite reductase.

Nitrite reductase [NAD(P)H] subunit. EC number (for enzymes) ; edit table. The effect of cyanide and ferricyanide on the activity of the dissimilatory nitrate reductase of Escherichia coli.

Biol. Chem.,– () PubMed Google Scholar [25] Aoki, K.; Shinke, R.; Nishira, H.: Isolation and identification of respiratory nitrate reductase-producing bacteria. The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli.

Biol. Chem.Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen. The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified fold from the cytoplasmic membrane.

Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of52, The enzyme contained molybdenum (mol/mol complex), iron (mol/mol. Unlike the heme cd 1 ‐based nitrite reductase enzymes, the molecular mechanism of copper‐containing nitrite reductases remains controversial.

A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small.

The physiological role of the periplasmic nitrate reductase, Nap, one of the three nitrate reductases synthesized by Escherichia coli K, has been investigated. Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon.

Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC ). The Bacterial Nitrate Reductase: EPR Studies on the Enzyme A of Escherichia coli K12, Biochim Biophys Acta74, • Enoch, H., and Lester, R.: The Role of a Novel Cytochrome b-Containing Nitrate Reductase and Quinone in the in Vitro Reconstitution of Formate-Nitrate-Reductase Activity of E.

coli, Biochem Biophys Res Com Purification and Spectropotentiometric Characterization of Escherichia coli NrfB, a Decaheme Homodimer That Transfers Electrons to the Decaheme Periplasmic Nitrite Reductase.

Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli K Purification of a hexaheme cytochrome c from Escherichia coli K12 and its properties as a nitrite reductase. not a hexahaem but a kDa tetrahaem nitrite reductase. The properties of PII were essentially similar to those of PII.

A preparatian of PII' with less ability to reduce trimethylamine N-oxide was obtained from another active peak obtained by Sephadex filtration. Nitrite reductase [EC ] was eluted in a different position from the enzyme reducing N-oxide.

In Escherichia coli, nitrosative mutagenesis may occur during nitrate or nitrite respiration. The endogenous nitrosating agent N2O3 (dinitrogen trioxide, nitrous anhydride) may be formed either by the condensation of nitrous acid or by the autooxidation of nitric oxide, both of which are metabolic by-products.

The purpose of this study was to determine which of these two agents is more. Escherichia coli contains a versatile respiratory chain which oxidizes ten different electron donor substrates and transfers the electrons to terminal reductases or oxidases for the reduction of six different electron acceptors.

Salmonella is able to use even two more electron acceptors. The variation is further increased by the presence of isoenzymes for some substrates. Guy D. Fauque, Larry L. Barton, in Advances in Microbial Physiology, Cytochrome c Nitrite Reductase from D. desulfuricans ATCC The dissimilatory reduction of nitrate and/or nitrite to ammonia (also called dissimilatory ammonification) can serve as the sole energy-conserving process in some species of e is reduced to ammonia (with nitrite as intermediate) by a few.Sulfite reductase (SiR) has been purified to homogeneity from spinach leaves.

Two forms of the enzyme were separated by hydroxylapatite chromatography. One, with subunit Mr 69 appears to be proteolytically cleaved to give rise to the other, with subunit Mr 63during the purification procedu .Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus.

Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components.

E. coli also expresses two membrane-bound proton-translocating nitrate .

2250 views Monday, November 30, 2020