scholarly journals Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration

2016 ◽  
Vol 92 (5) ◽  
pp. fiw034 ◽  
Author(s):  
Clément Bardon ◽  
Franck Poly ◽  
Florence Piola ◽  
Muriel Pancton ◽  
Gilles Comte ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Allosteric Transition ◽  
Biological Denitrification ◽  
Membrane Bound

scholarly journals Membrane-Bound Nitrate Reductase Is Required for Anaerobic Growth in Cystic Fibrosis Sputum

2007 ◽  
Vol 189 (12) ◽  
pp. 4449-4455 ◽  
Author(s):  
Kelli L. Palmer ◽  
Stacie A. Brown ◽  
Marvin Whiteley
Keyword(s):  
Cystic Fibrosis ◽  
Nitrate Reductase ◽  
Mutational Analysis ◽  
Autosomal Recessive Disorder ◽  
Opportunistic Pathogen ◽  
Anaerobic Growth ◽  
Expression Of Genes ◽  
Membrane Bound ◽  
Lung Infections

ABSTRACT The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aeruginosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aeruginosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF lung.

scholarly journals Periplasmic nitrate reduction in Wolinella succinogenes: cytoplasmic NapF facilitates NapA maturation and requires the menaquinol dehydrogenase NapH for membrane attachment

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2784-2794 ◽  
Author(s):  
Melanie Kern ◽  
Jörg Simon
Keyword(s):  
Electron Transport ◽  
Nitrate Reductase ◽  
Deletion Mutant ◽  
Electron Flow ◽  
Gene Clusters ◽  
Nitrate Respiration ◽  
Wolinella Succinogenes ◽  
Periplasmic Nitrate Reductase ◽  
Iron Sulfur ◽  
Membrane Bound

Various nitrate-reducing bacteria produce proteins of the periplasmic nitrate reductase (Nap) system to catalyse electron transport from the membraneous quinol pool to the periplasmic nitrate reductase NapA. The composition of the corresponding nap gene clusters varies but, in addition to napA, genes encoding at least one membrane-bound quinol dehydrogenase module (NapC and/or NapGH) are regularly present. Moreover, some nap loci predict accessory proteins such as the iron–sulfur protein NapF, whose function is poorly understood. Here, the role of NapF in nitrate respiration of the Epsilonproteobacterium Wolinella succinogenes was examined. Immunoblot analysis showed that NapF is located in the membrane fraction in nitrate-grown wild-type cells whereas it was found to be a soluble cytoplasmic protein in a napH deletion mutant. This finding indicates the formation of a membrane-bound NapGHF complex that is likely to catalyse NapH-dependent menaquinol oxidation and electron transport to the iron–sulfur adaptor proteins NapG and NapF, which are located on the periplasmic and cytoplasmic side of the membrane, respectively. The cysteine residues of a CX3CP motif and of the C-terminal tetra-cysteine cluster of NapH were found to be required for interaction with NapF. A napF deletion mutant accumulated the catalytically inactive cytoplasmic NapA precursor, suggesting that electron flow or direct interaction between NapF and NapA facilitated NapA assembly and/or export. On the other hand, NapA maturation and activity was not impaired in the absence of NapH, demonstrating that soluble NapF is functional. Each of the four tetra-cysteine motifs of NapF was modified but only one motif was found to be essential for efficient NapA maturation. It is concluded that the NapGHF complex plays a multifunctional role in menaquinol oxidation, electron transfer to periplasmic NapA and maturation of the cytoplasmic NapA precursor.

Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus).

2008 ◽  
Vol 55 (4) ◽  
pp. 753-760 ◽  
Author(s):  
Władysław Polcyn
Keyword(s):  
Mass Spectrometry ◽  
Nitrate Reductase ◽  
Biochemical Properties ◽  
Validation Criteria ◽  
Sds Page ◽  
Mass Spectrometry Identification ◽  
Bradyrhizobium Sp ◽  
Rhizobial Species ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.

scholarly journals Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

2006 ◽  
Vol 72 (8) ◽  
pp. 5173-5180 ◽  
Author(s):  
Helen Ridley ◽  
Carys A. Watts ◽  
David J. Richardson ◽  
Clive S. Butler
Keyword(s):  
Nitrate Reductase ◽  
Reductase Activity ◽  
Selective Reduction ◽  
Enterobacter Cloacae ◽  
Anaerobic Growth ◽  
Elemental Selenium ◽  
Membrane Bound ◽  
Membrane Bound Enzymes ◽  
Distinct Membrane

ABSTRACT Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ∼2 mM, with an observed V max of 500 nmol SeO4 2− min−1 mg−1 (k cat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.

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