ENGINEERING OF PYRIDINE-NUCLEOTIDE SPECIFICITY OF NITRATE REDUCTASE -MUTAGENESIS OF RECOMBINANT CYTOCHROME-B REDUCTASE FRAGMENT OF NEUROSPORA-CRASSA NADPH-NITRATE REDUCTASE

The cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a H is-tag for purification after mutation of the NADPH binding site, The recombinant enzyme fragment was altered by site-directed mutagenesis g uided by the three-dimensional structure of cytochrome b reductase fra gment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N, crassa) greatly increased preference for NADH, This mutant had n early the same NADH:ferricyanide reductase k(cat) as wildtype with NAD PH, Substitutions for Arg921 had little influence on coenzyme specific ity, while substitution of Ser or Gin for Arg932 did. The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had lo w activity and low affinity for coenzymes, which indicated a general l oss of specificity in the binding site, Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH, Wild t ype had a specificity ratio of 1100, which was defined as the catalyti c efficiency (k(cat)/K-m)for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the spec ificity ratio was reversed by over 6000-fold by a single mutation, Pre ference for NADPH versus NADH is strongly influenced by presence/absen ce of a negatively charged amino acid side chain in the binding site f or 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). (C) 1998 Academic Press.