Purification and partial characterization of glutamate synthase from <i>Rhodospirillum rubrum</i> grown under nitrogen-fixing conditions

Carlberg, Inger; Nordlund, Stefan

doi:10.1042/bj2790151

Cited by 16 publications

(23 citation statements)

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“…Pyridine-nucleotide-dependent glutamate synthase is present in higher plants , fungi (Hummlet and Mora, 1980), yeasts (Masters and Meister, 1982;Cogoni et al, 1995), algae (Calvin et al, 1984) and bacteria (Miller and Standtman, 1972;Ratti et al, 1985 ;Carlberg and Nordlund, 1991). Fd-dependent glutamate synthase is a monomeric protein of 130-170 kDa containing flavin and an iron-sulfur centre in higher plants (Hirasawa et al, 1996), algae (Galvin et al, 1984) and cyanobacteria (MarquCs et al, 1992).…”

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confidence: 99%

Structure and Regulation of Ferredoxin‐Dependent Glutamase Synthase from Arabidopsis Thaliana

Suzuki

Rothstein

1997

European Journal of Biochemistry

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Ferredoxin (Fd)-dependent glutamate synthase is present in green leaves, etiolated leaves, shoots and roots of Ambidopsis thaliana (ecotype Columbia). In photosynthetic green leaves and shoots, Fd-dependent glutamate synthase accounts for more than 96% of the total glutamate synthase activity in vitro with the remaining activity derived from an enzyme that uses NADH as the electron donor. In etiolated leaves and roots, Fd-dependent glutamate synthase is 3 -4-fold less active than in green leaves, but represents 70-85 % of the total glutamate synthase activity in these tissues. Fd-dependent glutamate synthase is detected as a single peptide of 165 kDa on a western blot of green leaf and shoot tissues, and this Fddependent glutamate synthase polypeptide is 3 -4-fold less abundant in etiolated leaves and roots. In these non-photosynthetic tissues, there is a higher activity of NADH-dependent glutamate synthase. The A. thaliana gltS mutant (strain CS254) contains only 1.7% and 17.5% of the wild-type Fd-dependent glutamate synthase activity in leaves and roots, respectively. Western blots indicate that the Fd-dependent glutamate synthase peptide of 165 kDa is absent from leaves and roots of the gltS mutant. In contrast, NADH-dependent glutamate synthase activity in leaves and roots is unaffected. During illumination of wild-type dark-grown leaves for 72 h, the levels of Fd-dependent glutamate synthase protein and its activity increased threefold to levels equivalent to those in green leaves. In contrast, NADH-dependent glutamate synthase activity decreases twofold during illumination. The complete nucleotide sequence of the complementary DNA for A. tlzaliana Fd-dependent glutamate synthase has been determined. Analysis of the amino acid sequence deduced from the complete cDNA sequence (5178 bp) has revealed that A. thaliana Fd-dependent glutamate synthase i s synthesized as a 1648-amino-acid precursor protein (I80090 Da) which consists of a 131-amino-acid transit peptide (14603 Da) and a 1517-amino-acid mature peptide (165487 Da). The A. thaliuria Fd-dependent glutamate synthase has a high similarity to maize Fd-dependent glutamate synthase (83 9%) and to the analogous region of NADH-dependent glutamate synthase (42 %) and NADPH-dependent glutamate synthases (40-43 %) from different organisms. The A. thuliaiiu Fd-dependent glutamate synthase contains the purF-type glutamine-amido-transfer domain as well as flavin and iron-sulfur-cluster-binding domains. The deduced primary structures of A. thaliaiza Fd-dependent glutamate synthase and of glutamate synthases from other organisms indicate that Fd-dependent glutamate synthase may have evolved from bacterial NADPH-dependent glutamate synthase. The cDNA hybridized to RNA of about 5.3 kb from different tissues of A. thaliana. A high steadystate level of Fd-dependent glutamate synthase mRNA is found in photosynthetic green leaves and shoots, and roots contain less mRNA for Fd-dependent glutamate synthase. In the gEtS mutant, there are twofold and fourfold lower levels of Fd...

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confidence: 99%

Structure and Regulation of Ferredoxin‐Dependent Glutamase Synthase from Arabidopsis Thaliana

Suzuki

Rothstein

1997

European Journal of Biochemistry

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“…GOGATs from bacteria are iron-sulphur flavoproteins, with the exception of that of Clostridium pasteurianum, which apparently possesses a different quaternary structure and lacks iron and flavin as prosthetic groups (Singhal et al, 1989). There have been only a few reports containing partial characterizations of GOGAT from phototrophic bacteria (Yelton & Yoch, 1981 ;Caballero et al, 1989b;Carlberg & Nordlund, 1991).…”

Section: Introductionmentioning

confidence: 99%

“…GOGAT from Rsp. rubrum has been proposed as being involved in regulating nitrogenase activity (Carlberg & Nordlund, 1991). R. capsulatus E l F l is a moderate halophilic diazotroph whose nitrogenase, nitrate reductase and GS seem to be controlled by C/N status (Moreno-Vivian et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

“…This subunit also contains flavin and non-haem iron in E. coli and seems to catalyse NH,+-dependent L-glutamate synthesis. In addition, bacterial GOGATs exhibit several aggregation states depending on ionic strength (Miller & Stadtman, 1972;Yelton & Yoch, 1981;Carlberg & Nordlund, 1991). In E. coli, two structural genes, gltB and gltD, form an operon with a regulatory gene, gltF (Castaii0 et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

“…GOGAT has been purified and partially characterized only from Rhodospirillum rubrum (Yelton & Yoch, 1981;Carlberg & Nordlund, 1991), a phototrophic bacterium which is not very closely related to R. capsulatus as deduced from 16s rRNA sequence analysis (Woese, 1987). GOGATs from heterotrophic bacteria are well-studied, dimeric enzymes of similar structural properties, quaternary structure and subunit size (Miller & Stadtman, 1972;Trotta et al, 1974;Schreier & Bernlohr, 1984;Ratti et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

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Molecular and kinetic characterization of glutamate synthase from the phototrophic bacterium Rhodobacter capsulatus E1F1

Igeño

Caballero

Castillo

1993

Journal of General Microbiology

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Glutamate synthase (GOGAT) from the phototrophic non-sulphur purple bacterium Rhodobactev capsulatus ElFl has been purified to electrophoretic homogeneity by affinity chromatography. The native protein consisted of two different subunits of 175 and 53 kDa and contained 4 mol FAD, 4 mol iron and 4 mol labile sulphide per mol of dimer enzyme. The enzyme used NADPH as the electron donor and was inhibited by iron-chelating and thiol group reagents. GOGAT exhibited NAD(P)H-diaphorase activity which used sodium ferricyanide, cytochrome c and dichlorophenol indophenol as alternative electron acceptors. By contrast, glutaminase activity was not detected in purified GOGAT. The amino acid composition was quite different from that of other bacterial GOGATs, and the protein presented different aggregation states depending on the ionic strength. Two major multimeric active species with Stokes' radii of 6-18 and 7.32 nm could be separated by gel-filtration of protein solutions made in 0.5 M-KCI, whereas in the absence of salt, the maximal GOGAT activity corresponded to an oligomer with Stokes' radius of 6.80 nm. The enzyme exhibited apparent negative cooperativity for glutamine, and was competitively inhibited by L-glutamate and NADP+.

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Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase

Filetici

Martegani

Valenzuela

et al. 1996

Yeast

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Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions

Cited by 16 publications

References 43 publications

Structure and Regulation of Ferredoxin‐Dependent Glutamase Synthase from Arabidopsis Thaliana

Structure and Regulation of Ferredoxin‐Dependent Glutamase Synthase from Arabidopsis Thaliana

Molecular and kinetic characterization of glutamate synthase from the phototrophic bacterium Rhodobacter capsulatus E1F1

Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase

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