title: Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase.
abstract: Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA. mbarber@hsc.usf.edu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Barber
authors.forename: Shawn K
authors.initials: SK
authors.lastname: Desai
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Marohnic
authors.forename: Hector H
authors.initials: HH
authors.lastname: Hernandez
authors.forename: Veronica V
authors.initials: VV
authors.lastname: Pollock
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003067:Coenzymes; D003574:Cytochrome c Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D050895:Nitrate Reductase (NADH); D009565:Nitrate Reductases / Q000096:biosynthesis* / Q000235:genetics*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011621:Pteridines / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018724:Spinacia oleracea / Q000201:enzymology; D015003:Yeasts
chemical_list: D003067:Coenzymes; D003574:Cytochrome c Group; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050895:Nitrate Reductase (NADH); D009565:Nitrate Reductases; D011621:Pteridines; C022261:molybdopterin