Assimilatory nitrate reductase: Lysine 741 participates in pyridine nucleotide binding via charge complementarity

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cyt ochrome b(557)-, and FAD-containing protein, catalyzes the regulated and ra te-limiting step in the utilization of inorganic nitrogen by high plants. W ith a recombinant, histidine-tagged form of the spinach nitrate reductase f lavin domain, site-directed mutagenesis has been utilized to examine the ro le of lysine 741 in binding the reducing substrate, NADH. Seven individual mutants, corresponding to K741R, K741H, K741A, K741E, K741M, K741Q, and K74 1P, have been engineered and six of the resulting proteins purified to homo geneity. With the exception of K741P, all the mutants were obtained as func tional flavoproteins which retained FAD as the sole prosthetic group and ex hibited spectroscopic properties comparable to those of the wild-type domai n, indicating that the amino acid substitutions had no effect on FAD bindin g. In contrast, all the mutants were found to have altered NADH:ferricyanid e reductase (NADH:FR) activity with mutations affecting both k(cat) and K-m (NADH), which decreased and increased, respectively. At pH 7.0, k(cat), dec reased in the order WT > K741R > K741A > K741H > K741E > K741M > K741Q whil e K-m(NADH) increased in the same order. The most efficient mutant, K741R, retained 80% of the wild-type NADH:FR activity, while in contrast the most inefficient mutant, K741Q, retained only 18% of the wildtype NADH:FR activi ty together with a 118-fold increased K-m(NAPH). pH studies of K741H reveal ed that both k(cat) and K-m(NAPH) were pH-dependent, with enhanced activity observed at acidic pH. These results indicated that retention of a positiv ely charged side chain at position 741 in the spinach nitrate reductase pri mary sequence is important for the efficient binding and subsequent oxidati on of NADH and that the positively charged side chain enhances nucleotide b inding via charge complementarity with the negatively charged pyrophosphate moiety. (C) 2001 Academic Press.