Shoji Ida

Purification and properties of ferredoxin—nitrate reductase from the cyanobacterium Plectonema boryanum

Abstract Assimilatory ferrodoxin—nitrate reductase has been purified to homogeneity from the cyanobacterium Plectonema boryanum . The enzyme was solubilized by sonication and, following heat treatment, ammonium sulfate precipitation and chromatography on DEAE-Toyopearl, Sephadex G-150 and hydroxyapatite, was purified 19000-fold with a yield of 15%. The purified enzyme had a specific activity of 305 and 1020 μmol NO 2 − formed/min per mg protein for ferrodoxin-linked and methyl viologen-linked nitrate reductase activities, respectively. The molecular weights measured by sodium dodecyl sulfate gel electrophoresis and sedimentation equilibrium were 83 000 and 85 000, respectively, indicating that the enzyme consists of a single polypeptide chain. The purified nitrate reductase contained 0.95 atom of molybdenum, and four iron and four acid-labile sulfur atoms per molecule ( M r 85 000). The spectrum of the enzyme resembled that of iron-sulfur proteins. The CD and MCD spectra suggested the presence of an iron-sulfur cluster in the molecule. Some similarities of amino-acid composition were observed between the cyanobacterial nitrate reductase, NAD(P)H-nitrate reductase and the catalytic subunit of the dissimilatory nitrate reductase from Escherichia coli . The stoichiometry of the nitrate reductase reaction confirmed a two-electron reduction of nitrate to nitrite by reduced ferredoxin or reduced methyl viologen. The K m values of the native enzyme was 38 μM for reduced ferredoxin and 2500 μM for reduced methyl viologen. Dithionite converted the native enzyme into a modified form which had no ferredoxin-linked nitrate reductase activity but retained methyl viologen-linked activity with a pH optimum at 10.2.

Isolation and characterization of NADH-glutamate synthase from pea (Pisum sativum L.).

Both ferredoxin-glutamate synthase (EC 1.4.7.1) and NADH-glutamate synthase (EC 1.4.1.14) were isolated separately on DEAE-cellulose chromatography from etiolated pea shoots. The latter enzyme was purified 1,400-fold by ammonium sulfate fractionation and column chromatographies of DEAE-cellulose, Sephadex G-200 and blue-Sepharose. The enzyme had a molecular weight of 220,000 and an isoelectric point of 4.3. The optimum pH was 7.6. Apparent Km values for l-glutamine, 2-oxoglutarate and NADH were 400, 37 and 4 µm, respectively. The enzyme had its absorption maxima at 275, 375 and 440 nm, suggesting that pea NADH-glutamate synthase is a flavoprotein. It showed NADH-diaphorase activity toward ferricyanide and 2,6-dichlorophenol indophenol as the electron acceptor. Sulfhydryl reagents, metal-chelating reagents, phthalein acids and azaserine were strong inhibitors. Ammonium and phosphate ions enhanced the enzyme activity.

Spinach ferredoxin-nitrite reductase: a purification procedure and characterization of chemical properties

Abstract An improved procedure for the purification of ferredoxin-nitrite reductase (ammonia:ferredoxin oxidoreductase, EC 1.7.7.1) from 18 kg of spinach leaves is described. The procedure involves acetone and ammonium sulfate fractionation, hydrophobic chromatography on benzyl- and phenyl-Sepharose and affinity chromatography on blue- and ferredoxin-Sepharose. The enzyme is homogeneous as judged by gel electrophoresis, sedimentation behavior and chemical analysis. The purified preparation shows methyl viologen-linked specific activity of 207 μmol NO2− reduced/min per mg protein. The purified enzyme shows absorption maxima at 280, 389, 574 and 690 nm with ϵ389 = 6.09 · 104 M−1·cm−1 and A389/A280 ratio of 0.65. The enzyme, Mr 63 000, contains 4.8 atoms Fe, of which 0.94 mol is siroheme, and 4.0 mol of acid-labile sulfide per molecule. The spinach ferredoxin-nitrite reductase consists of 558 amino acid residues, including four half-cysties and eight sulfhydryl groups. The NH2-terminal sequence is determined to be Ala-Val-Pro-Pro-Arg-Val-Asp-Ala-, while the COOH-terminal region is -Ala-Glu. Some hydrodynamic and kinetic properties are described.

Purification, stabilization and characterization of nitrite reductase from barley roots

SummaryNitrite reductase (NiR) isolated from barley (Hordeum vulgare L.) roots was stabilized in a buffer solution containing a sulfhydryl-reducing reagent and glycerol. The enzyme was purified 340fold by ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-cellulose. Purified NiR had a specific activity of 28 μmol NO2- reduced min-1 mg-1 of protein. The purified preparation was reddishbrown having absorption maxima at 282, 388 and 577 nm. The barley-root enzyme was almost identical with spinach-leaf NiR with respect to molecular weight, isoelectric point, pH stability, pH optimum, affinity for substrate, behavior toward inhibitors. It is concluded that NiR is the same enzymatic entity regardless of its localization in photosynthetic or nonchlorophyllous tissues. The electron-transport system for NiR in root tissue is discussed in comparison with that in leaf tissue.

Nucleotide sequence of a rice root ferredoxin-NADP+ reductase cDNA and its induction by nitrate.

A ferredoxin-NADP+ reductase (FNR) cDNA was isolated from a lambda gt 11 cDNA library constructed from the roots of nitrate-induced rice (Oryza sativa L. cv. Kinmaze) seedlings. The nucleotide sequence of this clone contains a 1134 nucleotide open reading frame. The N-terminal 62 amino acid stretch was assigned to the transit sequence, followed by 316 residues for the mature protein. The rice root FNR shows only 49% sequence identity to that of the leaf enzyme, but the regions of the binding sites to ferredoxin, NADP-PPi and NADP+ are highly conserved between the two enzymes. The root FNR mRNA was induced transiently by the addition of nitrate, but not by ammonia. The results support the view that the root FNR is involved in the nitrate assimilation in nonchlorophyllous tissues.

Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

Co-regulation of nitrate reductase and nitrite reductase in cultured spinach cells

Summary The analysis of nitrate reductase(NR)-deficient mutants provides an efficient approach to the study of the regulatory mechanisms of nitrate assimilation. We previously isolated two cell lines, 12F and I-1, and suggested that the mutation in the 12F cell line related to translation of NR mRNA which is expressed in the presence of nitrate, and that the mutation in the I-1 cell line may be in a regulatory gene controlling both genes encoding NR and nitrite reductase (NiR). We investigated transformants of the two cell lines using particle bombardment with tobacco NR cDNA and analyzed the expression of NR and NiR in the transformants. In the 12F cell line transformants, NR activity and protein was rescued completely and the transformants could grow on NO 3 - medium. This result indicates that the 12F cell line has a mutation that prevents the synthesis of NR protein from NR mRNA. In the I-1 cell line, the activities of NR and NiR were detected in cells grown on NO 3 - medium, but at low levels. In the transformants however, activities of NR and NiR and the NR mRNA levels attained levels of observed in the wild-type cells. In addition, the transformants could also grow on NO 3 - medium. These results indicate that the I-1 cell line has a mutation related to the signal transfer cascade from the NO 3 - ion to the NR gene. This suggests that NR and NiR genes are co-regulated, and that the presence of NR mRNA may be post-transcriptionally essential for the synthesis of NiR protein.

Ferredoxin-sepharose affinity chromatography for the purification of assimilatory nitrite reductase

Assimilatory nitrate reduction in higher plants and algae proceeds in two steps: the first stage is catalyzed by nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2.) and yields nitrate, and the subsequent step is catalyzed by nitrite reductase (ferredoxin-nitrite oxidoreductase, EC 1.6.6.4) to yield ammonia with the use of six electrons [1,2]. Recently nitrate reductase has been obtained in a highly purified state by the use of affinity chromatography in which FAD [3], Blue dextran [4], NADH [5], and p-nitroaniline, respectively [6] were used as specific or general ligands coupled to Sepharose gels. To date no attempts have been made to purify nitrite reductase (NiR) by affinity chromatography, although NiR has been prepared in a highly purified form by conventional purification procedures such as ion exchange chromatography, gel filtration and preparative gel electrophoresis [7], the yields of the enzyme were, however, rather low and the procedures were time-consuming. In this communication, we describe a simple and efficient method for the purification of NiR from a higher plant by application of ferredoxin-Sepharose affinity chromatography and some properties of the purified preparation.

Characterization of four molybdenum cofactor mutants of rice, Oryza sativa L.

Abstract Four nitrate reductase-deficient (NR − ) mutants, C25, C27, C32 and C33, were isolated from 100 000 M 2 seedlings of rice ( Oryza sativa L., ssp. indica , cv. IR30) by means of selection for chlorate-resistance. All mutations were monogenic and recessive. Biochemical analysis showed that both NADH- and NADPH-dependent activities of nitrate reductase (NR) in these four mutants were less than 25% of the wild type IR30. However, nitrite reductase activities were more than the wild type. Also, xanthine dehydrogenase (XDH) activities and molybdenum cofactor biosynthesis abilities (MoCo activities) were deficient indicating that these mutants were cnx and not nia type. The cnx mutants could be divided into two groups: one could recover NADH-NR activity by adding 0.5 mM molybdate to the growth medium (C27, C32 and C33) while the other could not (C25). The result of allelism test coincided with the phenotypic classification by adding molybdate. The growth response to tungstate was also investigated in the mutants. None of the mutants classifies to the tungstate-sensitive group as reported in Arabidopsis cnx mutants.

Characterization of a rice (Oryza sativa L.) mutant deficient in the heme domain of nitrate reductase

SummaryBiochemical and genetical characterization of a rice nitrate reductase (NR)-deficient mutant, M819, which had been isolated as a chlorate-resistant mutant, was carried out. In M819, leaf NADH-NR activity was found to be about 10% of that of the wild-type cv ‘Norin 8’, while NADPH-NR activity was higher than that in the wild-type; FMNH2-NR and MV-NR activities were also 10% of those of the wild type; BPB-NR activity was higher than that of the wild type; and xanthine dehydrogenase activity was revealed to be present in both. These results suggest that the mutant line M819 lacks the functional heme domain of the NADH-NR polypeptide due to a point mutation or a small deletion within the coding region of the structural gene. Chlorate resistance in M819 was transmitted by a single recessive nuclear gene.