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)

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 Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.
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PMID:Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli. 879 83

Direct electrochemical studies, utilizing two voltammetric methods-square-wave voltammetry (SWV) and cyclic voltammetry (CV)-have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+ analogs. The reduction potentials (E degrees ') of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2 as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25 degrees C), the two-electron reduction potential for the FAD/FADH2 couple has been determined to be -274 and -257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be -234 and -227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+ analogs have been determined to be as follows: NAD+ (E degrees ' = -192 mV), 5'-ADP ribose (E degrees ' = -199 mV), ADP (E degrees ' = -154 mV), AMP (E degrees ' = -196 mV), adenosine (E degrees ' = -192 mV), adenine (E degrees ' = -220 mV), and NMN (E degrees ' = -208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E degrees ' = -268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+ to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+ analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.
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PMID:Direct electrochemistry of the flavin domain of assimilatory nitrate reductase: effects of NAD+ and NAD+ analogs. 928 15