Induction of Nitrate Assimilatory Enzymes in the Tree <i>Betula pendula</i>

Friemann, Andreas; Lange, Michael; Hachtel, Wolfgang; Brinkmann, Klaus

doi:10.1104/pp.99.3.837

Cited by 25 publications

(15 citation statements)

References 25 publications

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“…Crude leaf extracts and partially purified NR were prepared as described earlier (Friemann et al, 1992). NR assays were performed in vitro in the presence of NADH and/or NADI'H in saturating concentrations (0.7 mM) by ineasuring the amount of nitrite formed in NR assay buffer at 25°C within 1 h after the addition of NR preparations (Hageman and Reed, 1980).…”

Section: In Vitro Assays Of Nrmentioning

confidence: 99%

The Choice of Reducing Substrate Is Altered by Replacement of an Alanine by a Proline in the FAD Domain of a Bispecific NAD(P)H-Nitrate Reductase from Birch

Schöndorf¹,

Hachtel²

1995

Plant Physiol.

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Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Befula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. l h e mutated cDNAs were integrated into the genome of tobacco by Agrobacferium-mediated transformation. Transgenic tobacco (Nicotiana fabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine8" by proline) is critical for the use of reducing substrate.

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Section: In Vitro Assays Of Nrmentioning

confidence: 99%

The Choice of Reducing Substrate Is Altered by Replacement of an Alanine by a Proline in the FAD Domain of a Bispecific NAD(P)H-Nitrate Reductase from Birch

Schöndorf¹,

Hachtel²

1995

Plant Physiol.

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“…Die fur PiIzc kennzeichnende NADPH-spczifischc N R kommt offcnbar nicht in hiihcren Pflanzen vor. Nur zwei Pflanzcnarten sind bckannt, die ausschliefllich iiber einc bispezifischc N R vcrfiigen; eine ist die von uns untcrsuchte Birke Betuh pendula [6,7] …”

Section: Bispezif Itatunclassified

Die Nitratreduktase höherer Pflanzen

Schöndorf

Hachtel

1994

Biologie in unserer Zeit

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“…The first, niaD3000, incorporated the putative NADHspecific ' DQ ' sequence from corn. The other two, niaD3001 and niaD3002, combined these mutations with changes to the prolines described earlier (Friemann et al, 1991 ;Garde et al, 1995 ;Shondorf & Hachtel, 1995). The plasmids were transformed into A. nidulans and the genomic DNA of a single transformant from each transformation was extracted and Southern-blotted to ensure each contained a single homologous integration of the pUC5hniiA\niaD plasmid.…”

Section: Production and Characterization Of Mutants With Altered Pyrimentioning

confidence: 99%

“…Higher plants usually contain an NADH-specific NaR (EC 1;6;6;1), which has also been found in some algae. A third type, NAD(P)H-bispecific NaR (EC 1;6;6;2), has been found in some higher plants and algae, but is rarely the sole NaR in these organisms (Friemann et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

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Structure–function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo

Hall

Tomsett

2000

Microbiology

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Nitrate reductase (NaR) catalyses the reduction of nitrate to nitrite via a twoelectron transfer. In fungi, the electron donor for NaR is NADPH whereas plants can have two enzymes, NADH :NaR and a bispecific NAD(P)H :NaR. PCR mutagenesis was employed to introduce mutations into the niaD gene of Aspergillus nidulans in order to identify residues involved in co-enzyme specificity. The niaD3000 mutation (NiaD T813D, K814Q) altered co-enzyme specificity : the new enzyme had high levels of NADH :NaR activity in vitro, whilst all NADPH-associated activity was lost. However, strains carrying this mutation did not grow on nitrate. Enzyme assays suggested that this was not due to inhibition of the mutant enzyme by NADPH. All revertants of the niaD3000 mutants had restored NADPH activity and lost NADH activity. Sequence analysis of these revertants showed that they all contained a single amino acid change at Asp-813, suggesting that this position is crucial to coenzyme specificity. Further studies have shown that the mutant enzyme was not protected from deactivation by either co-factor in cell-free extracts (unlike the wild-type), and that induction of the glucose-6-phosphate dehydrogenase occurred independently of NADPH levels. These data highlight the importance of functional tests in vivo under physiological conditions.

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Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula

Abstract: The coordinate appearance of the bispecific NAD(P)H-nitrate reductase (NR; EC 1.6.6.2) and nitrite reductase (NiR; EC 1.7.7

Cited by 25 publications

References 25 publications

The Choice of Reducing Substrate Is Altered by Replacement of an Alanine by a Proline in the FAD Domain of a Bispecific NAD(P)H-Nitrate Reductase from Birch

The Choice of Reducing Substrate Is Altered by Replacement of an Alanine by a Proline in the FAD Domain of a Bispecific NAD(P)H-Nitrate Reductase from Birch

Die Nitratreduktase höherer Pflanzen

Structure–function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo

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