Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.
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PMID:Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli. 702 35

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.
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PMID:The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase. 730 57

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.
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PMID:The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha. 763 19

Three genes, narH, narJ and narI, of the membrane-bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced. The derived gene products show high sequence similarity to the equivalent (beta, putative delta and gamma) subunits of the two membrane-bound nitrate reductases of the enteric bacterium Escherichia coli. All iron-sulphur cluster ligands proposed for the E. coli beta subunits are conserved in T. pantotropha NarH. Secondary structure analysis of NarJ suggests that this protein has a predominantly alpha-helical structure. Comparison of T. pantotropha NarI with the b-haem-binding integral membrane subunits of the E. coli enzymes allows assignment of His-53, His-63, His-186 and His-204 (T. pantotropha NarI numbering) as b-haem axial ligands and the construction of a three-dimensional model of this subunit. This model, in which the two b-haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems. Similar movement of electrons is required in the membrane-bound uptake hydrogenases and membrane-bound formate dehydrogenases. We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer. One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate-reducing bacteria has structural resemblance to NarI.
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PMID:Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop. 774 53

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 the E. coli chromosome. The narGHJI operon, encoding nitrate reductaseA, is located in the chlC locus at 27 minutes, along with several functionally related genes: narK, encoding a nitrate/nitrite antiporter, and the narXL operon, encoding a nitrate-activated, two component regulatory system. The narZYWV operon, encoding nitrate reductase Z, is located in the chlZ locus located at 32.5 minutes, a region which includes a narK homologue, narU, but no apparent homologue to the narXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both the narGHJI operon and the narK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while the narZYWV operon and the narU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase in E. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.
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PMID:Nitrate reductases in Escherichia coli. 774 40

A strain of Pseudomonas putida that can express a nitrate reductase that is located in the periplasmic compartment was isolated from freshwater. The enzyme was active in vivo during arginine fermentation and at the onset of oxygen limitation in batch cultures. The activity of the enzyme increased the yield of bacteria following fermentative growth under anoxic conditions with arginine, but nitrate reduction did not support growth on non-fermentable carbon substrates under anoxic conditions. Cells expressing the periplasmic nitrate reductase were capable of reducing nitrate in the presence of oxygen. Nitrate reduction under oxic conditions was clearly coupled to a respiratory electron transport chain because: (1) the process was sensitive to the respiratory inhibitors rote-none and 2-n-heptyl-4-hydroxyquinoline N-oxide, and (2) membrane-bound and periplasmic cytochromes were involved. This is the first report of the presence of a periplasmic nitrate reductase in a member of the gamma proteobacteria.
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PMID:Isolation and characterisation of a strain of Pseudomonas putida that can express a periplasmic nitrate reductase. 777 73

A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphaera pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrificans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of T. pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed.
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PMID:Mo(V) electron paramagnetic resonance signals from the periplasmic nitrate reductase of Thiosphaera pantotropha. 781 68

The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGHJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumarate reductase (frdABCD) operons among others. The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression. To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified. Both proteins were autophosphorylated in the presence of [gamma-32P]ATP and MgCl2 but not with [alpha-32P]ATP. Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate. In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate. In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.
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PMID:Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli. 805 Oct 11

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.
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PMID:Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha. 811 78

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.
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PMID:Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity. 812 39


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