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

Nitrate reductase (NaR) catalyses the reduction of nitrate to nitrite via a two-electron transfer. In fungi, the electron donor for NaR is NADPH where as 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 Asper gillus nidulans in order to identify residues involved in co-enzyme specifi city. The niaD3000 mutation (NiaD T813D, K814Q) altered co-enzyme specifici ty: the new enzyme had high levels of NADH:NaR activity in vitro, whilst al l NADPH-associated activity was lost. However, strains carrying this mutati on did not grow on nitrate. Enzyme assays suggested that this was not due t o inhibition of the mutant enzyme by NADPH, All revertants of the niaD3000 mutants had restored NADPH activity and lost NADH activity, Sequence analys is of these revertants showed that they all contained a single amino acid c hange at Asp-813, suggesting that this position is crucial to coenzyme spec ificity. Further studies have shown that the mutant enzyme was not protecte d from deactivation by either co-factor in cell-free extracts (unlike the w ild-type), and that induction of the glucose-6-phosphate dehydrogenase occu rred independently of NADPH levels. These data highlight the importance of functional tests in vivo under physiological conditions.