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 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 cytochrome b reductase fragment 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 nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln 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 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).
Shiraishi
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Phytotechnology Research Center and Department of Biological Sciences, Michigan Technological University, 1400 Townsend Drive, Houghton, Michigan, 49931-1295, USA.
Croy
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Kaur
J
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Campbell
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003579:Cytochrome Reductases / Q000096:biosynthesis / Q000235:genetics*; D042966:Cytochrome-B(5) Reductase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology*; D050897:Nitrate Reductase (NADPH); D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D011742:Pyrimidine Nucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics
D010446:Peptide Fragments; D011742:Pyrimidine Nucleotides; D011994:Recombinant Proteins; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D009565:Nitrate Reductases; D050897:Nitrate Reductase (NADPH)