scholarly journals The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha

1995 ◽  
Vol 309 (3) ◽  
pp. 983-992 ◽  
Author(s):  
B C Berks ◽  
D J Richardson ◽  
A Reilly ◽  
A C Willis ◽  
S J Ferguson
Keyword(s):  
Nitrate Reductase ◽  
Sequence Similarity ◽  
Alcaligenes Eutrophus ◽  
Integral Membrane Protein ◽  
Periplasmic Nitrate Reductase ◽  
Thiosphaera Pantotropha ◽  
Cysteine Motif ◽  
Membrane Bound ◽  
K 12 ◽  
Nitrate Reductases

The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.

scholarly journals Catabolite Repression Control of napF (Periplasmic Nitrate Reductase) Operon Expression in Escherichia coli K-12

2008 ◽  
Vol 191 (3) ◽  
pp. 996-1005 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe ◽  
Li-Ling Chen ◽  
Amie Cai
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Nitrate Reductase ◽  
Catabolite Repression ◽  
Carbon Sources ◽  
Anaerobic Growth ◽  
Periplasmic Nitrate Reductase ◽  
Membrane Bound ◽  
Operon Expression ◽  
K 12

ABSTRACT Escherichia coli, a facultative aerobe, expresses two distinct respiratory nitrate reductases. The periplasmic NapABC enzyme likely functions during growth in nitrate-limited environments, whereas the membrane-bound NarGHI enzyme functions during growth in nitrate-rich environments. Maximal expression of the napFDAGHBC operon encoding periplasmic nitrate reductase results from synergistic transcription activation by the Fnr and phospho-NarP proteins, acting in response to anaerobiosis and nitrate or nitrite, respectively. Here, we report that, during anaerobic growth with no added nitrate, less-preferred carbon sources stimulated napF operon expression by as much as fourfold relative to glucose. Deletion analysis identified a cyclic AMP receptor protein (Crp) binding site upstream of the NarP and Fnr sites as being required for this stimulation. The napD and nrfA operon control regions from Shewanella spp. also have apparent Crp and Fnr sites, and expression from the Shewanella oneidensis nrfA control region cloned in E. coli was subject to catabolite repression. In contrast, the carbon source had relatively little effect on expression of the narGHJI operon encoding membrane-bound nitrate reductase under any growth condition tested. Carbon source oxidation state had no influence on synthesis of either nitrate reductase. The results suggest that the Fnr and Crp proteins may act synergistically to enhance NapABC synthesis during growth with poor carbon sources to help obtain energy from low levels of nitrate.

scholarly journals Characterization of the Reduction of Selenate and Tellurite by Nitrate Reductases

2001 ◽  
Vol 67 (11) ◽  
pp. 5122-5126 ◽  
Author(s):  
Monique Sabaty ◽  
Cécile Avazeri ◽  
David Pignol ◽  
André Vermeglio
Keyword(s):  
Nitrate Reductase ◽  
Ralstonia Eutropha ◽  
Paracoccus Denitrificans ◽  
Potassium Tellurite ◽  
Benzyl Viologen ◽  
Periplasmic Nitrate Reductase ◽  
Membrane Bound ◽  
Nitrate Reductases

ABSTRACT Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V max andKm were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 μmol · min−1 · mg−1 and 0.12 mM were obtained for V max and Km , respectively, whereas the V max values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.

scholarly journals Structural investigation of the molybdenum site of the periplasmic nitrate reductase from Thiosphaera pantotropha by X-ray absorption spectroscopy

1996 ◽  
Vol 317 (2) ◽  
pp. 557-563 ◽  
Author(s):  
Brian BENNETT ◽  
John M. CHARNOCK ◽  
Heather J. SEARS ◽  
Ben C. BERKS ◽  
Andrew J. THOMSON ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Absorption Spectroscopy ◽  
X Ray ◽  
Periplasmic Nitrate Reductase ◽  
Thiosphaera Pantotropha ◽  
Treated Sample ◽  
Membrane Bound ◽  
X Ray Absorption ◽  
Best Fit ◽  
Respiratory Nitrate Reductase

The molybdenum centre of the periplasmic respiratory nitrate reductase from the denitrifying bacterium Thiosphaera pantotropha has been probed using molybdenum K-edge X-ray absorption spectroscopy. The optimum fit of the Mo(VI) EXAFS suggests two =O, three –S– and either a fourth –S– or an –O–/–N– as molybdenum ligands in the ferricyanide-oxidized enzyme. Three of the –S– ligands are proposed to be the two sulphur atoms of the molybdopterin dithiolene group and Cys-181. Comparison of the EXAFS of the ferricyanide-oxidized enzyme with that of a nitrate-treated sample containing 30% Mo(V) suggests that the Mo(VI) → Mo(V) reduction is accompanied by conversion of one =O to –O–. The best fit to the Mo(IV) EXAFS of dithionite-reduced enzyme was obtained using one =O, one –O– and four –S–/–Cl ligands. The periplasmic nitrate reductase molybdenum co-ordination environment in both the Mo(VI) and Mo(IV) oxidation states is distinct from that found in the membrane-bound respiratory nitrate reductase.

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.

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

Evolution of the soluble nitrate reductase: defining the monomeric periplasmic nitrate reductase subgroup

2006 ◽  
Vol 34 (1) ◽  
pp. 122-126 ◽  
Author(s):  
B.J.N. Jepson ◽  
A. Marietou ◽  
S. Mohan ◽  
J.A. Cole ◽  
C.S. Butler ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Structural Alignment ◽  
Bioinformatic Analysis ◽  
Catalytic Subunit ◽  
Surface Polarity ◽  
Redox Partner ◽  
Redox Partners ◽  
Membrane Bound ◽  
And Function ◽  
Nitrate Reductases

Bacterial nitrate reductases can be classified into at least three groups according to their localization and function, namely membrane-bound (NAR) or periplasmic (NAP) respiratory and cytoplasmic assimilatory (NAS) enzymes. Monomeric NASs are the simplest of the soluble nitrate reductases, although heterodimeric NASs exist, and a common structural arrangement of NAP is that of a NapAB heterodimer. Using bioinformatic analysis of published genomes, we have identified more representatives of a monomeric class of NAP, which is the evolutionary link between the monomeric NASs and the heterodimeric NAPs. This has further established the monomeric structural clade of NAP. The operons of the monomeric NAP do not contain NapB and suggest that other redox partners are employed by these enzymes, including NapM or NapG predicted proteins. A structural alignment and comparison of the monomeric and heterodimeric NAPs suggests that a difference in surface polarity is related to the interaction of the respective catalytic subunit and redox partner.

scholarly journals Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

1998 ◽  
Vol 180 (16) ◽  
pp. 4192-4198 ◽  
Author(s):  
Andrew J. Darwin ◽  
Eva C. Ziegelhoffer ◽  
Patricia J. Kiley ◽  
Valley Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Binding Site ◽  
Anaerobic Growth ◽  
Periplasmic Nitrate Reductase ◽  
Operon Expression ◽  
Nitrate And Nitrite ◽  
K 12

ABSTRACT The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, thenapF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napFcontrol region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

The periplasmic nitrate reductase of Thiosphaera pantotropha

1995 ◽  
Vol 59 (2-3) ◽  
pp. 728
Author(s):  
Ben C. Berks ◽  
Brian Bennett ◽  
Jacques Breton ◽  
Ann Reilly ◽  
Anthony C. Willis ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Periplasmic Nitrate Reductase ◽  
Thiosphaera Pantotropha

scholarly journals The Periplasmic Nitrate Reductase inPseudomonas sp. Strain G-179 Catalyzes the First Step of Denitrification

1999 ◽  
Vol 181 (9) ◽  
pp. 2802-2806 ◽  
Author(s):  
Laura Bedzyk ◽  
Tao Wang ◽  
Rick W. Ye
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Primary Role ◽  
Anaerobic Conditions ◽  
It Use ◽  
Alternative Substrate ◽  
Periplasmic Nitrate Reductase ◽  
Reduction Activity ◽  
Membrane Bound

ABSTRACT Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase inPseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap− mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.

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