Glutamate synthase from Bacillus subtilis PCI 219

Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources.     

Glutamate synthase in rice roots. Studies on the electron donor specificity

Rice root glutamate synthase activity was assayed with various reducing systems. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and pyridine nucleotide-dependent glutamate synthase (NADH, EC 1.4.1.14; or NADPH, EC 1.4.1.13) exhibited a strict specificity for the electron donor. The ferredoxin-dependent glutamate synthase from rice roots could accept electrons from photoreduced ferredoxin in an illuminated reconstituted spinach chloroplast system. Thioredoxin, a potent electron carrier, was not able to provide either ferredoxin-dependent or pyridine nucleotide-dependent glutamate synthase with electrons as no glutamate formation was detected in the presence of reduced thioredoxin f or m.     

Cysteine synthase from rape leaves.

Cysteine synthase [O-Acetyl-L-serine acetate-lyase (adding hydrogen-sulfide) EC 4.2.99.8] has been highly purified from the extract of rape, Brassica chinensis var. Komatsuna. The purified preparation appeared to be homogeneous on Sephadex G-100 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis, showing a molecular weight of about 62,000. The latter method also suggested that this enzyme was composed of two identical subunits. The enzyme contained 2 moles of pyridoxal phosphate per mole of enzyme.     

Characterization of flavone synthase I from rice

Flavones are synthesized from flavanones through the action of flavone synthases (FNSs). There are two FNSs, FNS I and II. FNS I is a soluble dioxygenase present in members of the Apiaceae family and FNS II is a membrane bound cytochrome P450 enzyme that has been identified in numerous plant species. In this study, we cloned OsFNS I-1 from rice by RTPCR, expressed it in E. coli, and purified the recombinant protein. By NMR analysis, we found that OsFNS I-1 converted the flavanone (2S)-naringenin into the flavone, apigenin. Moreover, we found that the cofactors oxoglutarate, FeSO(4), ascorbate and catalase are required for this reaction. OsFNS I-1 encodes a flavone synthase I. This is the first type I FNS I found outside of the Apiaceae family.     

Inactivation of nitrate reductase from wheat and rice leaves

In fresh leaves, the inactivation of nitrate reductase was rapid at high temperatures as compared to low temperatures. In leaves subjected to freeze-thaw treatment, the loss of enzyme activity was extremely rapid particularly at high temperatures. Pre-incubation with NADH not only protected the enzyme against inactivation, but also stimulated its activity. In dialysed extracts of rice leaves, NADH alone offered some protection while nitrate alone did not protect the enzyme from inactivation. Addition of both NADH and nitrate during pre-incubation enhanced the enzyme activity considerably. It is suggested that stimulation of nitrate reduction by NADH and nitrate may be of physiological significance to the plant, in the sense that in the presence of sufficient supplies of reluctant and nitrate, the process of nitrate assimilation would be accelerated.     

Purification and characterization of caffeine synthase from tea leaves.

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 microM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.     

A protocol for high-throughput extraction of DNA from rice leaves

Current DNA isolation methods have limitations between speed and purity in high-throughput molecular genetic analysis such as gene mapping and marker-assisted selection programs. We have optimized a simple and rapid method for isolating high-quality genomic DNA from rice that significantly minimizes time and the use of laboratory materials. One person can process as many as 384 samples in 2 h. The isolated DNA is suitable for polymerase chain reaction-based techniques and is stable for no less than 6 mo of storage at 4°C.     

Antibacterial diterpenes and their fatty acid conjugates from rice leaves

Six structurally oryzalide-related compounds, oryzadione (1), 2, 3, 4, 5 and 6, were isolated from a neutral fraction of the extract of healthy leaves using a bacterial leaf blight-resistant cultivar of a rice plant, "Norin-27", as a group of antimicrobial substances. Their structures were determined by spectroscopic studies to be kaurane analogues and kaurane analogues conjugated with fatty acids, i.e., 1: ent-15,16-epoxy-kauran-2,3-dione (enol form: ent-15,16-epoxy-2-hydroxy-kauran-1-en-3-one), 2: ent-15,16-epoxy-3beta-hydroxy-kauran-2-one, 3: ent-15,16-epoxy-3-oxa-kauran-2-one, 4: ent-15,16-epoxy-3beta-myristoyloxy-kauran-2-one, 5: ent-15,16-epoxy-3alpha-palmitoyloxy-kauran-2-one, and 6: ent-15,16-epoxy-2beta-palmitoyloxy-kauran-2-one.     

Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues

Rabbit antiserum was raised against ferredoxin-dependent glutamate synthase (EC 1.4.7.1) purified from green leaves of Oryza sativa L. cv Delta. Ferredoxin-dependent glutamate synthase, detected in green leaf, etiolated leaf, and root tissues cross-reacted completely with the antiferredoxin glutamate synthase immunoglobulin G. In contrast, the immunoglobulin G did not cross-react with NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases found in nonphotosynthetic etiolated leaf and root tissues. In addition, ferredoxin-dependent glutamate synthase was separated and distinguished by its affinity to ferredoxin from NAD(P)H-dependent glutamate synthase on ferredoxin-Sepharose affinity chromatography. Based on the immunological studies, it is suggested that ferredoxin-dependent glutamate synthases in green leaf and etiolated leaf tissues are closely related proteins; in contrast, ferredoxin-dependent glutamate synthase in root tissue is a distinct protein from the leaf enzymes.     

适于蛋白双向电泳的水稻叶片样品提取方法初探

在水稻基因组测序完成后,利用蛋白质组学技术揭示水稻基因功能的研究,已成为水稻分子生物学研究的热点之一。水稻叶片作为DNA研究的便利材料被经常使用,但对蛋白质研究来说,占叶片全蛋白50%～60%的核酮糖二磷酸羧化酶（RuBP羧化酶）对低丰度蛋白常常造成掩盖。以水稻叶片为材料,用不同浓度的聚乙二醇（PEG）去除叶片中RuBP羧化酶。通过SDS-PAGE垂直电泳比较发现,浓度为17%的PEG对去除RuBP羧化酶效果最好,所获得的蛋白质样品可以得到质量较高的双向电泳图谱。     

Glutamate synthase in greening callus of Bouvardia ternifolia Schlecht

The distribution of the two glutamate-synthase (GOGAT) activities known to exist in higher plants (NADH dependent, EC 2.6.1.53; and ferredoxin dependent, EC 1.4.7.1) was studied in non-chlorophyllous and chlorophyllous cultured tissue as well as in young leaves of Bouvardia ternifolia. The NADH-GOGAT was present in all three tissues. Using a sucrose gradient we found it in both the soluble and the plastid fraction of non-chlorophyllous and chlorophyllous tissue, but exclusively in the chloroplast fraction of the leaves. Ferredoxin-GOGAT was found only in green tissues and was confined to the chloroplasts. Ferredoxin-GOGAT activity increased in parallel with the chlorophyll content of the callus during the greening process in Murashige-Skoog medium (nitrate and ammonium as the nitrogen sources), while NADH-GOGAT was not affected by the greening process in this medium. Furthermore, both activities were differentially affected by either nitrate or ammonium as the sole nitrogen source in the medium during this process. It is suggested that each GOGAT activity is a different entity or is differently regulated.Abbreviations GOGAT glutamate synthase - MS Murashige-Skoog (1962) medium - PMSF phenylmethylsulfonyl fluoride     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Methionine-induced phytoalexin production in rice leaves

The application of methionine on wounded rice leaves induced the production of rice phytoalexins, sakuranetin and momilactone A. This induction resulted from stimulation of phenylalanine ammonia-lyase and naringenin 7-O-methyltransferase activity. Jasmonic acid, ethylene, and active oxygen species are important as signal transducers in disease resistance mechanisms. However, although the endogenous level of jasmonic acid rapidly increased in reaction to wound, methionine treatment could not induced endogenous JA production. Ethylene induced the production of the flavonoid phytoalexin, sakuranetin, but did not induce the production of a terpenoid phytoalexin, momilactone A. On the other hand, a free radical scavenger, Tiron, counteracted the induction of both sakuranetin and momilactone A production in methionine-treated leaves. Active oxygen species may be important in methionine-induced production of phytoalexins.     

Induced volatiles in elicitor-treated and rice blast fungus-inoculated rice leaves

The volatiles released from elicitor (copper chloride, jasmonic acid, UV, L-methionine and chitosan oligomer)-treated and rice blast fungus-inoculated rice leaves were collected by the solid-phase microextraction technique and analyzed by GC-MS. (Z)-3-Hexen-1-ol, monoterpenes, methyl salicylate, and sesquiterpenes were identified as elicitor-induced volatiles by a comparison of their GC retention times and mass spectra with those of authentic compounds. The different elicitors resulted in some qualitative and quantitative differences in the production of volatiles. Monoterpenes and sesquiterpenes were identified as the rice blast fungus-induced volatiles.     

Glutamate synthase from Chlamydomonas reinhardtii: Interaction studies with its substrate ferredoxin and molecular cloning

Glutamate synthase (GOGAT) from Chlamydomonas reinhardtii is able to form functional covalent complexes with its substrate ferredoxin (Fd), either wild-type (^WTFd) or recombinant form (^rFd). However, when Fd carboxyl groups were chemically modified (^mdFd), no complexes were detected and its ability to serve as electron donor for glutamate synthase activity was also decreased. By site-directed mutagenesis, we have demonstrated that Fd glu91 and a negative core in the helix α1 are critical for Fd interaction with this enzyme and its functionality as electron carrier for glutamate synthase. As a previous step to elucidate the specific positive charged residues involved in glutamate synthase interaction with Fd, we have isolated a cDNA, CrFG-3, encoding Fd-GOGAT from C. reinhardtii. The cDNA comprised about 60% of the protein and sequence comparison showed that CrFG-3 was structurally more similar to higher plant enzymes than to the corresponding prokaryotic GOGAT. Two conserved domains were present in this protein fragment, the FMN-binding domain and the cysteines involved in the iron–sulfur cluster binding. This revised version was published online in June 2006 with corrections to the Cover Date.