Engineering of Pyridine Nucleotide Specificity of Nitrate Reductase: Mutagenesis of Recombinant CytochromebReductase Fragment ofNeurospora crassaNADPH:Nitrate Reductase

The cytochromebreductase fragment ofNeurospora crassaNADPH:nitrate reductase (EC 1.6.6.3) was overexpressed inEscherichia coliwith a His-tag for purification after mutation of the NADPH binding site. The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochromebreductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase ofN. crassa) greatly increased preference for NADH. This mutant had nearly the same NADH:ferricyanide reductasekcatas wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did. The cytochromebreductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site. Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH. Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the specificity ratio was reversed by over 6000-fold by a single mutation. Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2′ phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2).