1887

Abstract

Nitrate reductase (NaR) catalyses the reduction of nitrate to nitrite via a two-electron 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 gene of in order to identify residues involved in co-enzyme specificity. The mutation (NiaD T813D, K814Q) altered co-enzyme specificity: the new enzyme had high levels of NADH:NaR activity , 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 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 co-enzyme 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 under physiological conditions.

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2000-06-01
2020-01-29
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