Metabolism of [6-14C]orotate by shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus

Incorporation of radioactivity from [6-¹⁴C]orotate into the pyrimidine constituents of shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus was examined with special reference to the unusual pyrimidine constituents. With each species, although 80% of the orotate supplied was catabolized to β-alanine, all the pyrimidine derivatives became radioactively labelled. With Pisum, the major part of the radioactivity incorporated into pyrimidines was located in UMP and the uracil derivatives, including the uracilyl amino acids willardiine and isowillardiine. With Phaseolus, UMP and the uracil derivatives were again the major radioactive products; incorporation of radioactivity into 5-ribosyluracil (pseudouridine), which accumulates in Phaseolus tissues, was comparable to the incorporation into orotidine and twice that found in cytidine. Lathyrus incorporated a substantially larger part of the presented [6-¹⁴C] orotate into pyrimidine derivatives than did the other two species. CMP was the most highly radioactive product, followed next by lathyrine and UMP. Surprisingly, 20% of the total radioactivity incorporated into pyrimidines by Lathyrus was located in the pyrimidine amino acid lathyrine. This confirms previous evidence that lathyrine is essentially a product of the orotate pathway. The overall recovery of radioactivity in all three species was 93–95%. The data emphasize the necessity of including the less common pyrimidine constituents, as well as the common ones, in quantitative studies of pyrimidine metabolism in plants.     

Structure of the S1S2 glutamate binding domain of GLuR3

Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. Determining the structural differences between the binding sites of different subtypes is crucial to our understanding of neuronal circuits and to the development of subtype specific drugs. The structures of the binding domain (S1S2) of the GluR3 (flip) AMPA receptor subunit bound to glutamate and AMPA and the GluR2 (flop) subunit bound to glutamate were determined by X‐ray crystallography to 1.9, 2.1, and 1.55 Å, respectively. Overall, the structure of GluR3 (flip) S1S2 is very similar to GluR2 (flop) S1S2 (backbone RMSD of 0.30 ± 0.05 for glutamate‐bound and 0.26 ± 0.01 for AMPA‐bound). The differences in the flip and flop isoforms are subtle and largely arise from one hydrogen bond across the dimer interface and associated water molecules. Comparison of the binding affinity for various agonists and partial agonists suggest that the S1S2 domains of GluR2 and GluR3 show only small differences in affinity, unlike what is found for the intact receptors (with the exception of one ligand, Cl‐HIBO, which has a 10‐fold difference in affinity for GluR2 vs. GluR3). Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Free amino acids and γ-glutamyl peptides in seeds of Fagus silvatica

The contents of amino acids and peptides have been investigated in seeds of Fagus silvatica L. (beechnuts). In addition to the common amino acids, the following compounds have been isolated and identified: 4-hydroxyproline (probably the cis-l-isomer), N⁵-acetylornithine, 3-(2-furoyl)-l-alanine, methionine sulfoxide (probably an artefact), pipecolic acid (probably partially racemized d-isomer), l-willardiine (with a small amount of the d-isomer), N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid, N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid, 2(S),5(S),6(S)-5-hydroxy-6-methylpipecolic acid, 2(S),5(R),6(S)-5-hydroxy-6-methylpipecolic acid, γ-glutamylalanine, γ-glutamylglutamic acid, γ-glutamylisoleucine, γ-glutamylleucine, γ-glutamylmethionine sulfoxide (probably an artefact), γ-glutamylphenylalanine, γ-glutamyltyrosine, γ-glutamylvaline, glutathione, γ-glutamylwillardiine, and γ-glutamylphenylalanylwillardiine. γ-Glutamylphenylalanine and willardiine are the dominating components of the amino acid fraction.The isolations were performed by use of ion exchange chromatography, taking advantage of the different pK-values of the amino acids, mainly on acid resins in the 3-chloropyridinium form with aq. 3-chloropyridine as eluant and on basic resins in the acetate form with aqueous acetic acid as eluant. These methods in combination with preparative paper chromatography have permitted the isolation and identification of compounds present in amounts as low as 1/6000 of the dominant ninhydrin-reactive component. The implications of the occurrence of this large variety of compounds in the Fagaceae are briefly discussed.     

Partial purification and properties of willardiine and isowillardiine synthase activity from Pisum sativum

The enzymic activity responsible for synthesis of willardiine and isowillardiine in pea seedlings has been extracted and partially purified. Fresh tissue, pulverized in liquid-N₂, was extracted in a phosphate buffer (pH 7) and subjected to fractional precipitation with ammonium sulphate. After desalting on Sephadex G-25 and concentration by ultrafiltration, the fraction containing the activity was chromatographed sequentially on DEAE-Sepharose CL-6B, DEAE-cellulose (DE 52) and Sephadex G-200. Electrophoretic separation in polyacrylamide gels was also used. A 120-fold purification was achieved but at no stage was there any indication of a separation of willardiine synthase activity from that of isowillardiine synthase. Both activities paralleled one another when the enzymic preparation was progressively denatured by subjecting it to gradually increasing temperatures. Similarly, ageing at 4° and at ?196° resulted in a parallel loss of activity. Both synthase activities were maximal at 7.8–7.9and the pH optimum curves were of closely similar shape. From the results described, it is concluded that a single enzyme of relatively low MW (ca 50 000) is responsible for the synthesis of both uracilylalanines. Studies of the alanylation of uracil using a pyridoxal-metal ion model-enzyme system are described.     

γ-Glutamylwillardiine and γ-glutamylphenylalanylwillardiine from seeds of Fagus silvatica

γ-l-Glutamyl-l-willardiine [γ-l-glutamyl-3-(1-uracil)-l-l-alanine] and γ-glutamylphenylalanylwillardiine have been isolated from seeds of Fagus silvatica. The structures have been established by spectroscopy, hydrolysis to give the constituent amino acids, and for the tripeptide end-group determination and partial hydrolysis to give the two constituent dipeptides.     

Biosyntheses of the uracilylalanines willardiine and isowillardiine in higher plants

The syntheses of willardiine and isowillardiine were studied in vivo in pea seedlings by feeding uracil-[2-¹⁴C] and in vitro with enzyme extracts from Pisum, Lathyrus, Albizia, Leucaena and Fagus seedlings by using uracil and O-acetyl-l-serine as substrates. The fate of isowillardiine in the intact seedlings has also been studied by feeding isowillardiine-[¹⁴C] through the roots and determining its distribution. Some properties of willardiine and isowillardiine synthase(s) are described. Willardiine was also obtained by a B₆-catalysed chemical reaction under mild conditions. The question whether two enzymes are involved in the formation of the two isomeric uracilylalanines is discussed.