HORT640 - Metabolic Plant Physiology
Nitrate uptake and reduction
Nitrate and nitrite reductase structures
Ferredoxin-nitrite reductase (NiR) [EC 188.8.131.52] catalyzes the six-electron reduction of nitrite to ammonia, using reduced ferredoxin (Fd) as the electron donor. The Fd-dependent NiRs of plants, algae and cyanobacteria are monomeric proteins with molecular masses near 63 kDa which contain a single [4Fe-4S] cluster and a single siroheme (which serves as the binding site for nitrite) as prosthetic groups (Dose et al, 1997). Fd-NiR has been cloned from several higher plant sources, including Betula verrucosa, spinach and maize. Note that the plant enzyme is distinct from the NAD(P)H-nitrite reductase of bacteria and fungi [EC 184.108.40.206].
The six-electron reduction of sulfite to sulfide resembles that of the reduction of nitrite to ammonia (Crane and Getzoff, 1996). Both sulfite and nitrite reductases share an unusual prosthetic assembly in their active centers, namely siroheme covalently linked to an 4Fe-4S cluster (Crane and Getzoff, 1996).
Siroheme, is a cofactor of both sulfite and nitrite reductase in Salmonella typhimurium, and requires the cysG gene for its synthesis (Goldman and Roth, 1993). Three steps are required to synthesize siroheme from uroporphyrinogen III (a methylated, iron-containing modified tetrapyrrole), the last common intermediate in the heme and siroheme pathways. In Salmonella, cysG mutants are defective in the synthesis of cobalamin (B12), which shares a common precursor with siroheme (Goldman and Roth, 1993). A cysG mutant strain of Rhizobium etli that is pleiotropically defective in sulfate and nitrate assimilation has a mutation in a siroheme synthetase-homologous gene (Tate et al, 1997).
A cDNA (UPM1) was cloned from Arabidopsis thaliana that functionally complements an Escherichia coli cysG mutant that is unable to catalyze the conversion of uroporphyrinogen III to siroheme (Leustek et al, 1997). UPM1 encodes a 369-amino acid, 39.9-kDa protein. The UPM1 product has a sequence at the amino terminus that resembles a transit peptide for localization to mitochondria or plastids. The protein produced by in vitro expression is able to enter isolated intact chloroplasts but not mitochondria (Leustek et al, 1997). The UPM1 product contains two regions that are identical to consensus sequences found in bacterial uroporphyrinogen III and precorrin methyltransferases (Leustek et al, 1997). Recombinant UPM1 protein catalyzes S-adenosyl-L-methionine-dependent transmethylation by UPM1 (Leustek et al, 1997).
A full-length cDNA clone (pZmSUMT1) encoding an S-adenosyl-L-methionine-dependent uroporphyrinogen III C-methyltransferase (SUMT) [EC 220.127.116.11] has also been isolated from a maize root cDNA library (Sakakibara et al, 1996). The deduced amino acid sequence of the open reading frame of the cDNA is similar to that of SUMT from various bacteria and also to the SUMT catalytic region of siroheme synthase (cysG) from Escherichia coli. Overproduction of ZmSUMT1 in a cysG mutant of E. coli eliminated the requirement of the strain for cysteine (Sakakibara et al, 1996). The gene is induced in both roots and leaves in response to the addition of nitrate to the culture medium; the gene product is imported into plastids. ZmSUMT1 might be involved in the synthesis of siroheme, a prosthetic group of both nitrite and sulfite reductase. Gene expression is co-regulated with that of other nitrate-assimilatory genes (Sakakibara et al, 1996).
Nitrate induces a novel ferredoxin isoprotein in maize roots. It is proposed that this novel Fd-isoprotein might play an important role as an electron carrier from NADPH to NiR and other Fd-dependent enzymes in root plastids (Matsumura et al, 1997). NADPH may support nitrite reduction via a Fd-NADP+ oxidoreductase (FNR) (Jin et al, 1998).
Note the requirement for molybdenum (Mo) in the activity of nitrate reductase (NR) [EC 18.104.22.168]. NR in higher plants is proposed to be a homo-dimer, with two identical subunits joined and held together by the molybdenum cofactor (MoC or MoCo) (subunit molecular weight = 100 - 114 kDa) (Kleinhofs et al, 1989). The structure of the molybdenum cofactor is described by Rajagopalan (1989).
NR has two "hinge" regions situated between the co-factor binding regions. Hinge 1 between MC-NR (the molybdenum-containing nitrate-reducing fragment) and Cyt b contains a site where trypsin can access the backbone. Hinge 2 between the Cyt b binding region and the cytochrome b reductase fragment of NR also may contain a protease site.
A cysteine residue (Cys-191) in the molybdo-pterin region of the Arabidopsis NIA2 nitrate reductase protein is essential for enzyme activity (Su et al, 1997). It is proposed that this Cys residue provides a ligand to molybdenum in the MoCo-binding region.
Crane BR, Getzoff ED 1996 The relationship between structure and function for the sulfite reductases. Curr. Opin. Struct. Biol. 6: 744-756.
Dose MM, Hirasawa M, Kleis-SanFrancisco S, Lew EL, Knaff DB 1997 The ferredoxin-binding site of ferredoxin:nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin. Plant Physiol. 114: 1047-1053.
Goldman BS, Roth JR 1993 Genetic structure and regulation of the cysG gene in Salmonella typhimurium. J. Bacteriol. 175: 1457-1466.
Jin T, Huppe HC, Turpin DH 1998 In vitro reconstitution of electron transport from glucose-6-phosphate and NADPH to nitrite. Plant Physiol. 117: 303-309.
Kleinhofs A, Warner RL, Melzer JM 1989 Genetics and molecular biology of higher plant nitrate reductases. In (JE Poulton, JT Romeo, EE Conn eds) "Plant Nitrogen Metabolism", Recent Advances in Phytochemistry, Vol 23, Plenum Press, New York, pp. 117-155.
Leustek T, Smith M, Murillo M, Singh DP, Smith AG, Woodcock SC, Awan SJ, Warren MJ 1997 Siroheme biosynthesis in higher plants. Analysis of an S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase from Arabidopsis thaliana. J. Biol. Chem. 272: 2744-2752.
Matsumura T, Sakakibara H, Nakano R, Kimata Y, Sugiyama T, Hase T 1997 A nitrate-inducible ferredoxin in maize roots: genomic organization and differential expression of two nonphotosynthetic ferredoxin isoproteins. Plant Physiol. 114: 653-660.
Rajagopalan KV 1989 Chemistry and biology of the molybdenum cofactor. In (JL Wray, JR Kinghorn eds) "Molecular and Genetic Aspects of Nitrate Assimilation", Oxford Science Publications, Oxford, pp. 212-227.
Sakakibara H, Takei K, Sugiyama T 1996 Isolation and characterization of a cDNA that encodes maize uroporphyrinogen III methyltransferase, an enzyme involved in the synthesis of siroheme, which is prosthetic group of nitrite reductase. Plant J. 10: 883-892.
Su W, Mertens JA, Kanamaru K, Campbell WH, Crawford NM 1997 Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris. Plant Physiol. 115: 1135-1143.
Tate R, Riccio A, Iaccarino M, Patriarca EJ 1997 A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation. J. Bacteriol. 179: 7343-7350.
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