Abstract
The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.
Similar content being viewed by others
References
Akins RA, Lambowitz AM (1985) General method for cloning Neurospora crassa nuclear genes by complementation of mutants. Mol Cell Biol 5:2272–2278
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Bradford MM (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Broyles RH, Pack BM, Berger S, Dorn AR (1979) Quantitation of small amounts of hemoglobin in polyacrylamide gels with benzidine. Anal Biochem 94:211–219
Burger G, Strauss J, Scazzocchio C, Lang F (1991) nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions. Mol Cell Biol 11:5746–5755
Campbell WH, Kinghorn JR (1990) Functional domains of assimilatory nitrate reductase and nitrite reductases. Trends Biochem Sci 15:315–19
Cherniack AD, Gárriga G, Kittle JD, Lambowitz AM (1990) Function of Neurospora mitochondrial tyrosyl tRNA-synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases. Cell 62:745–755
Cove DJ, Pateman JA (1969) Autoregulation of the synthesis of of nitrate reductase in Aspergillus nidulans. J Bacteriol 97:1374–1378
Davis RH, deSerres F (1970) Genetic and microbial research techniques for Neurospora crassa. Methods Enzymol 17A:79–143
Ebbole D, Sachs MS (1990) A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia. Fungal Genet Newslett 37:17–18
Fu YH, Marzluf GA (1987) Molecular cloning and analysis of the regulation of Nit-3, the structural gene for nitrate reductase in Neurospora crassa. Proc Natl Acad Sci USA 84:8243–8247
Fu Y, Marzluf GA (1988) Metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of Neurospora crassa. J Bacteriol 170:657–661
Garrett RH, Cove DJ (1976) Formation of NADPH-nitrate reductase activity in vitro from Aspergillus modulans maD and enx mutants. Mol Gen Genet 149:179–186
Garrett RH, Nason A (1967) Involvement of a b-type cytochrome in the assimilatory nitrate reductase of Neurospora crassa. Proc Natl Acad Sci USA 58:1603–1610
Garrett RH, Nason A (1969) Further purification and properties of Neurospora nitrate reductase. J Biol Chem 244:2870–2882
Jarai G, Marzluf GA (1991) Sulfate transport in Neurospora crassa: regulation, turnover, and cellular localization of the CYS-14 protein. Biochemistry 30:4768–4773
Johnstone IL, McCabe PC, Greaves P, Gurr SJ, Cole GE, Brow MAD, Unkles SE, Clutterbuck AJ, Kinghorn JR, Innis MA (1990) Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene 90:181–192
Karplus PA, Daniels MJ, Herriott JR (1991) Atomic structure of ferrodoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. Science 251:60–66
Kudla B, Caddick MX, Langdon T, Martinez-Rossi NM, Bennett CF et al. (1990) The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9:1355–64
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685
Lê KHD, Lederer F (1983) On the presence of a heme-binding domain homologous to cytochrome b5 in Neurospora crassa assimilatory nitrate reductase. EMBO J 2:1909–1914
Leach J, Finkelstein DB, Rambosek JA (1986) Rapid miniprep of DNA from filamentous fungi. Neurospora Newslett 33:32–33
Lin S, Dunn JJ, Studier FW, Stafford DW (1987) Expression of human factor IX and its subfragments in Escherichia coli and generation of antibodies to the subfragments. Biochemistry 26:5267–5274
Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Marzluf GA (1981) Regulation of nitrogen metabolism and gene expression in fungi. Microbiol Rev 45:437–461
Mathews FS, Levine M, Argos P (1972) Three-dimensional Fourier synthesis of calf liver cytochrome b5 at 2.8 Å resolution. J Mol Biol 64:449–464
Metzenberg RL, Baisch TJ (1981) An easy method for preparing Neurospora DNA. Neurospora Newslett 28:20–21
Okamoto PM, Fu YH, Marzluf GA (1991) Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Mol Gen Genet 227:213–223
Okamoto PM, Garrett RH, Marzluf GA (1993) Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa. Mol Gen Genet 238:81–90
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Solomonson LP, Bargber MJ (1990) Assimilatory nitrate reductase: functional properties and regulation. Annu Rev Plant Physiol Plant Mol Biol 41:225–253
Sorger GJ, DeBanne MT, Davies J (1974) Effect of nitrate on the synthesis and decay of nitrate reductase of Neurospora. Biochem J 140:395–403
Studier FW, Moffatt BA (1966) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Tomsett AB, Garrett RH (1981) Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase. Mol Gen Genet 184:183–190
Yuan G, Fu YH, Marzluf GA (1991) nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain. Mol Cell Biol 11:5735–5745
Author information
Authors and Affiliations
Additional information
Communicated by W Gajewski
Rights and permissions
About this article
Cite this article
Okamoto, P.M., Marzluf, G.A. Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis. Molec. Gen. Genet. 240, 221–230 (1993). https://doi.org/10.1007/BF00277060
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00277060