Abstract
Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 − null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.
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Aliverti A, Piubelli L, Zanetti G, Lubberstedt T, Hermann RG, Curti B (1993) The role of cysteine residues of spinach ferrodoxin-NADP+ reductase as assessed by site-directed mutagenesis. Biochemistry 32:6374–6380
Andrews SC, Shipley C, Keen JN, Findlay JB, Harrison PM, Guest JR (1992) The haemoglobin-like protein (HMG) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferrodoxin NADP+ reductases. FEBS Lett 302:247–252
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–254
Campbell WH, Kinghorn JR (1990) Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends Biochem Sci 15:315–319
Correll CC, Batie CJ, Ballou CP, Ludwig ML (1992) Phtalate dehydrogenase reductase: a modular structure for electron transfer from pyridine nucleotides to [2Fe-2S]. Science 258:1604–1610
Crawford NM, Arst HN (1993) The molecular genetics of nitrate assimilation in fungi and plants. Annu Rev Genet 27:115–146
Davis RH, deSerres FJ (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol 17A:79–143
Dwivedi UN, Shiraishi N, Campbell WH (1994) Identification of an “essential” cysteine of nitrate reductase via mutagenesis of its recombinant cytochrome b reductase domain. J Biol Chem 269:13785–13791
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 YH, Marzluf GA (1990) nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proc Natl Acad Sci USA 87:5331–5335
Fu YH, Feng B, Evans S, Marzluf GA (1995) Sequence-specific DNA binding by NIT4, the pathway-specific regulatory protein that mediates nitrate induction in Neurospora. Mol Microbiol 15:935–942
Hyde GE, Campbell WH (1990) High level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH: cytochrome B5 reductase. Biochem Biophys Res Comm 168:1285–1291
Karplus PA, Daniles MJ, Herriott JR (1991) Atomic structure of ferrodoxin-NADP + reductase: prototype for a structurally novel flavoenzyme family. Science 251:60–66
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492
Okamoto PM, Marzluf GA (1993) Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis. Mol Gen Genet 240:221–230
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
Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for β-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci USA 85:6870–6874
Shirabe K, Yubisui T, Nishino T, Takeshita M (1991) Role of cysteine residues in human NADH: cytochrome b5 reductase studied by site-directed mutagenesis. J Biol Chem 266:7531–7536
Solomonson LP, Barber MJ, Robbins AP, Oaks A (1986) Functional domains of assimilatory NADH: nitrate reductase from Chlorella. J Biol Chem 261:11290–11294
Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130
Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci USA 83:4869–4873
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Communicated by C. A. M. J. J. van den Hondel
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González, C., Brito, N. & Marzluf, G.A. Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase. Molec. Gen. Genet. 249, 456–464 (1995). https://doi.org/10.1007/BF00287108
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DOI: https://doi.org/10.1007/BF00287108