Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

Okamoto, Patricia M.; Fu, Ying‐Hui; Marzluf, George A.

doi:10.1007/bf00259673

Cited by 79 publications

(53 citation statements)

References 47 publications

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“…4) is also a negative feedback loop, which, like in higher plants and algae, may be capable of acting as an autonomous daily rhythm generator. The inactivation and turnover of Neurospora NR and its mRNA have been found to be fairly rapid processes (Okamoto et al, 1991;Sorger et al, 1978;Walls et al, 1978). As for the FRQ negative feedback loop (Ruoff et al, 1999), the degradation of NR and its mRNA may play a role in defining the period length of the NR oscillator and its temperature behavior.…”

Section: Resultsmentioning

confidence: 99%

A Nitrate-Induced frq-Less Oscillator in Neurospora crassa

et al. 2004

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When nitrate is the only nitrogen source, Neurospora crassa's nitrate reductase (NR) shows endogenous oscillations in its nitrate reductase activity (NRA) on a circadian time scale. These NRA oscillations can be observed in darkness or continuous light conditions and also in a frq 9 mutant in which no functional FRQ protein is formed. Even in a white-collar-1 knockout mutant, NRA oscillations have been observed, although with a highly reduced amplitude. This indicates that the NRA oscillations are not a simple output rhythm of the whitecollar-driven frq oscillator but may be generated by another oscillator that contains the nit-3 autoregulatory negative feedback loop as a part. In this negative feedback loop, a product in the reaction chain catalyzed by nitrate reductase, probably glutamine, induces repression of the nitrate reductase gene and thus downregulates its own production. This is the first example of an endogenous, nutritionally induced daily rhythm with known molecular components that is observed in the absence of an intact FRQ protein.

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Section: Resultsmentioning

confidence: 99%

A Nitrate-Induced frq-Less Oscillator in Neurospora crassa

et al. 2004

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“…This repeat has also been noticed in the 5' untranslated region of the nit-3+ gene in N. crassa, although its significance remains unknown (18). The TACC repeat is followed by a purine-rich segment.…”

mentioning

confidence: 83%

Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase.

Alex

Borkovich

Simon

1996

Proc. Natl. Acad. Sci. U.S.A.

205

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“…To address the hypothesis that heterologously expressed P-450 systems in bacterial cells are limited by the availability of NADPH, we have constructed a series of reductase proteins designed to use NADH more efficiently. Specific mutations in the nicotinamide cofactor 2Ј-phosphate-binding site were generated based on the crystal structure of the reductase from rat (4) and from sequence alignments of rat cytochrome P-450 reductase (8) with other NADPH-and NADH-specific reductases including spinach ferredoxin-NADP ϩ reductase (9), nitrate reductases from Neurospora crassa (NADPH) (10) and corn (NADH) (11), and NADH-cytochrome b 5 reductase (12).…”

Section: Ferredoxin-nadpmentioning

confidence: 99%

Modification of the Nucleotide Cofactor-binding Site of Cytochrome P-450 Reductase to Enhance Turnover with NADH in Vivo

Elmore

Porter

2002

Journal of Biological Chemistry

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NADPH-cytochrome P-450 reductase is the electron transfer partner for the cytochromes P-450, heme oxygenase, and squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine has been shown to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP ؉ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently without inhibition by NADP ؉ . Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (k cat /K m ) with NADH. Inhibition by NADP ؉ was reduced by up to 4 orders of magnitude relative to the W677A protein and was equal to or less than that of the wildtype reductase. Both proteins were 2-3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/ W677A protein increased the sensitivity to dimethylnitrosamine by ϳ2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is the electron-donating partner for the cytochromes P-450, squalene monooxygenase, and heme oxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase is essential to cholesterol and steroid synthesis, and its importance is underscored by the developmental abnormalities and embryonic lethality observed in P-450 reductase-null mice (1). P-450 reductase contains 1 mol of FAD/ mol of enzyme and 1 mol of FMN/mol of enzyme. Reduced NADPH transfers two electrons as a hydride ion to the FAD of the enzyme, and FMN then accepts single electrons from the FAD and acts as the exit point to the protein acceptor such as the cytochromes P-450. P-450 reductase is a multi-domain protein, with the FAD and NADP(H) domains homologous with ferredoxin-NADP ϩ reductase and the FMN domain homologous with flavodoxin, suggesting that P-450 reductase was formed from an ancestral gene fusion event (2, 3). A fourth domain acts as a hinge to orient the two flavin-containing domains for electron transfer, and an NH 2 -terminal membrane-binding domain anchors the protein to the cytosolic side of the endoplasmic reticulum. The NADP(H) domain adopts the typical dinucleotide fold structure consisting of alternating ␣-helices and ␤-strands, with the nicotinamide cofactor binding in a cleft between the FAD and NADP(H) domains (4).Although expression of mammalian P-450/reductase systems in bacteria has been shown to be a valuable research tool and has promise as a tool for biodegradation (...

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Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

Cited by 79 publications

References 47 publications

A Nitrate-Induced frq-Less Oscillator in Neurospora crassa

A Nitrate-Induced frq-Less Oscillator in Neurospora crassa

Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase.

Modification of the Nucleotide Cofactor-binding Site of Cytochrome P-450 Reductase to Enhance Turnover with NADH in Vivo

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