Isamu Murakoshi

Enzymic synthesis of non-protein β-substituted alanines and some higher homologues in plants

Abstract Naturally occurring heterocyclic β -substituted alanines such as l -quisqualic acid and β -(pyrazol-1-yl)- l -alanine are enzymatically synthesized from O -acetyl- l -serine (OAS) and suitable precursors by cysteine synthases (CSase, O -acetylserine(thiol)lyase [EC 4.2.99.8]). By contrast, l -willardiine or the isomeric l -isowillardiine are synthesized by uracilylalanine synthases, in the same manner as the formation of l -cysteine as a primary metabolite. CSase isoenzymes also catalyse the formation of the neurotoxic amino acid gb -cyano- l -alanine from OAS and cyanide as an additional activity. This activity is different from the biosynthesis of β -cyano- l -alanine with l -cysteine and cyanide as a substrate, catalysed by β -cyanoalanine synthase (BCASase, [EC 4.4.1.9]) isolated from some higher plants. The properties of the purified CSase isoenzymes suggest that all CSases in higher plants are multifunctional enzymes involved in the biosynthesis of some β -substituted alanines, and that these enzymes may have different substrate specificities. Several properties, including the amino acid compositions and the immunological characterization of the purified CSases, are given. Furthermore the physiological role of these enzymes in higher plants is described. The biosynthesis in vitro of γ -substituted α -aminobutyric acids, which are the higher homologues of heterocyclic β -substituted alanines with one more carbon in the side chain, and the biomimetic synthesis of some heterocyclic β -substituted alanines catalysed by pyridoxal 5′-phosphate and metal ions, are also described.

cDNA cloning and expression of cysteine synthase B localized in chloroplasts of Spinacia oleracea

The cDNA clones for cysteine synthase B, which is localized in chloroplasts of Spinacia oleracea L., were isolated by screening a library with synthetic oligonucleotides encoding a partial peptide sequence of the purified protein. Nucleotide sequence analysis revealed an open reading frame encoding a polypeptide of 383 amino acids containing a putative transit peptide of 52 amino acids. A bacterial expression vector of the cDNA clone could genetically complement an Escherichia coli auxotroph lacking cysteine synthase and could produce the functionally active and immuno‐reactive cysteine synthase in E. coli. RNA blot hybridization suggested that the transcripts were primarily accumulated in leaves of spinach.

Purification and properties of cysteine synthase from Spinacia oleracea

Abstract Cysteine synthase was purified 3200-fold from Spinacia oleracea leaves. The purified enzyme has an apparent M , of 60 000 ± 2000 and can be dissociated into identical subunits of M , 32 000 ± 2000. The subunits contain one molecule of pyridoxal 5′-phosphate. The K m value is 2.9 mM for O -acetyl- L -serine and 22 μM for sulphide. Cysteine synthase from S. oleracea catalysed the formation of β-(pyrazol-1-yl)- L -alanine, and β-(3-amino-1,2,4-triazol-1-yl)- L -alanine, and significant differences were found between this enzyme and β-substituted alanine synthases and cysteine synthase from other sources. Amino acid composition of the purified enzyme was also determined.

Molecular cloning of a cysteine synthase cDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys- auxotroph.

We have isolated cDNA clones encoding cysteine synthase (CSase, EC 4.2.99.8), which catalyzes the terminal step in cysteine biosynthesis, by direct genetic complementation of a Cys− mutation in Escherichia coli with an expression library of Citrullus vulgaris (watermelon) cDNA. The library was constructed from 8-day-old etiolated seedlings of C. vulgaris in the λZAPII vector, converted to a plasmid library by in vivo excision, and then used for transformation of cysteine auxotroph E. coli NK3, which lacks the cysK and cysM loci. The complementing cDNA containing a 560 by 5′-untranslated region encodes a polypeptide of 325 amino acids of Mr 34342. The translational product reacted with an antibody raised against CSase A of Spinacia oleracea. CSase and β-pyrazolealanine synthase activities were demonstrated in vitro in extracts from E. coli cells expressing the cDNA. Genomic DNA blot analysis indicated the presence of a single copy of the gene, designated cysA, in the C. vulgaris genome. RNA blot hybridization indicated constitutive expression of cysA in cotyledons, hypocotyls and radicles of green and etiolated seedlings. These data suggested that this cDNA clone encodes CSase A the homolog of which in spinach is localized in the cytoplasm. The molecular phylogenetic tree of the amino acid sequences of CSaes from plants and bacteria suggested that there are three families in the CSase superfamily; the plant CSase A family, the plant CSase B family and the bacterial CSase family. The proteins in the plant CSase A family are the most conserved relative to the ancestral CSase protein.

Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

SummaryTransgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3′ end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R0 tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.

Two flavonol glycosides from seeds of Camellia sinensis

Two novel flavonol triglycosides, camelliaside A and B, have been isolated from seeds of Camellia sinensis. The structures were determined to be kaempferol 3-O-[2-O-beta-D- galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside and kaempferol 3-O-[2-O-beta- D-xylopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside on the basis of spectroscopic, chemical and enzymatic studies. These types of interglycosidic linkages, Gal(1----2)[Rha(1----6)]Glc and Xyl(1----2)[Rha(1----6)]Glc, have not been reported previously in flavone and flavonol glycosides.

Entadamide C, a sulphur-containing amide from Entada phaseoloides

Abstract A third new sulphur-containing amide, entadamide C, has been isolated from the leaves of Entada phaseoloides together with entadamide A. The stereostructure including the absolute configuration of entadamide C was established as (R)-(+)-trans-N-(2-hydroxyethyl)-3-methylsulphinylpropenamide, the sulphoxide form of entadamide A, by spectroscopic methods and physical properties. Chemical synthesis of (±)-entadamide C was achieved in three steps from propiolic acid.

Purification and characterization of cysteine synthases from Citrullus vulgaris

Abstract Two forms of cysteine synthase from Citrullus vulgaris seedlings were purified ca 2000-fold to homogeneity, respectively, using both conventional and affinity chromatographic methods. Both isoenzymes were found to have the same Mrs of58 000, and SDS-PAGE showed that they contained two identical subunits with Mrs of 29 000, respectively. Amino acid analysis indicated that isoenzymes A and B have almost similar amino acid compositions, except for the number of cysteine and methionine residues. The Km value of isoenzyme A is 2.6 mM for O-acetyl- l -serine (OAS) and 36 μM for sulphide, while that of isoenzyme B is 1.5 mM for OAS and 33 μM for sulphide. Data on the substrate specificity show that both isoenzymes catalyse the formation of β-(pyrazol-1-yl)- l -alanine and β-(3-amino-1,2,4-triazol-1-yl)- l -alanine, although only isoenzyme A catalyses the formation of β-cyano- l -alanine. Several properties of the purified cysteine synthase isoenzymes are described.

Genetic transformation of foxglove (Digitalis purpurea) by chimeric foreign genes and production of cardioactive glycosides

The chimeric neo and gus genes on a mini Ti vector are efficiently transferred into the genome of fox glove (Digitalis purpurea L.) using a binary vector system based on a rootinducing Ri plasmid, pRi15834. The transgenic state of established transformed roots was confirmed by Southern blot analysis and by detection of agropine and mannopine. The expression of the chimeric genes controlled by the promoters from TR 1′–2′ genes, nos gene and cauliflower mosaic virus 35S RNA was demonstrated by enzymatic and histochemical assays of neomycin phosphotransferase II and ß-glucuronidase. Enzyme-linked immunosorbent assay (ELISA) was carried out using polyclonal antibody reactable against digitoxin to investigate the production of cardenolides. The results of ELISA indicated that the cardioactive glycosides were highly produced in the green transformed hairy roots.

N-Formylcytisine: A new alkaloid from Thermopsis chinensis

Abstract A new base have been isolated from Thermopsis chinensis along with N-methylcytisine, cytisine, anagyrine and lupanine. The new alkaloid have been shown to be N-formylcytisine.