Conversion of hydroxylated and methylated dihydroflavonols into anthocyanins in a white flowering mutant of Petunia hybrida

A 3′, 4′-dihydroxy or a 3′, 4′, 5′-trihydroxy substitution pattern of dihydroflavonols is required for their conversion into the corresponding anthocyanins in a white flower of Petunia hybrida. The presence of a 5-hydroxyl group is not required. B-ring methylated dihydroflavonols were not converted into the corresponding anthocyanins. In case of a 4′-methoxy substituted dihydroflavonol a 4′-hydroxyanthocyanin is obtained, suggesting demethylation of this compound. The conversion of synthetic (±)-trans-2,3-dihydroflavonols into anthocyanins proceeded almost as well as with natural compounds. The results demonstrate that the cinnamic acid starter hypothesis for the origin of B-ring substituents is not correct for B-ring methylation.     

Der substitutionsspezifische einfluss von chalkonen auf die anthocyanbiosynthese bei Petunia hybrida

The effect of two chalcones, 3,4,2′,4′,6′-pentahydroxy- and the 4, 2 ′,4′,6′ -tetrahydroxy- 3-methoxy-chalcone- 4′-glucoside, on the synthesis of different flower anthocyanins in isolated petals of Petunia hybrida has been investigated. The results show that the synthesis of those anthocyanins, which have the same substitution pattern as the chalcone used was increased. This suggests that the chalcones are incorporated into the anthocyanins concerned. When the chalcones were fed together with acetic acid-[1-¹⁴C], this specific substitution effect was detectable only for the 3,4,2′,4′,6′-pentahydroxy-chalcone-4′-glucoside.     

Morphology and structure of γ-benzyl-l-glutamate copolymerized with l-phenylalanine, l-valine and l-alanine

Observation of random copolypeptides of γ-benzyl-l-glutamate with l-phenylalanine, l-valine and l-alanine was carried out in an electron microscope with samples cast from dilute solution. The relationship between the morphology and the molecular conformation in solution was studied with mixed solvents composed of chloroform and trifluoroacetic acid; these show a preference for α-helix and random coil, respectively. From the solutions in which molecules take α-helical conformation, fibrous films of nematic structure were formed. From random coil solutions discrete precipitates with folded molecules such as lamellar single crystals, piles of lamellae and structureless particles were formed. A copolypeptide containing l-valine in sufficiently large quantity to form β-structure also showed a variation in morphology with solvent, from films to discrete precipitates. It is suggested that the change in stiffness of the molecules contributes to the morphological variation.     

Methylation of anthocyanins by cell-free extracts of flower buds of Petunia hybrida

An O-methyltransferase activity which catalyses the methylation of anthocyanins was extracted from flowerbuds of Petunia hybrida. The methyltransferase uses S-adenosyl-l-methionine as methyl donor. Only anthocyanidin 3(p-coumaroyl)rutinosido-5-glucoside was methylated. No methylating activity towards anthocyanidins, anthocyanidin 3-glucosides, anthocyanidin 3-rutinosides, caffeic acid or p-coumaric acid could be detected.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

Biosynthesis of triterpenoids from amino acids in Pisum sativum. The distribution of the radioactivity in squalene biosynthesized from radioisotopically labelled l-leucine and l-valine

Radioisotopically labelled l-leucine and l-valine were fed to Pisum sativum and incorporated into squalene and β-amyrin. Chemical degradation of the radioactive squalene revealed an equal distribution of the radioactivity in the isopentenyl pyrophosphate(IPP)-derived and the 3,3-dimethylallyl pyrophosphate(DMAPP)-derived moieties of the squalene molecule, unlike the unbalanced distribution in favour of the DMAPP-derived moiety of a monoterpenoid molecule biosynthesized from these amino acids by higher plants.     

Pyrrolizidine alkaloid biosynthesis: Derivation of retronecine from l-arginine and l-ornithine

The relative retention of ³H and ¹⁴C on incorporation of d-, l- and dl-isomers of [¹⁴C]arginine and [¹⁴C]ornithine into retrorsine using L-[5-³H]arginine as an internal standard has been measured. The retronecine portion of the pyrrolizidine alkaloid retrorsine, present in Senecio isatideus plants, is shown to be derived from l-arginine and l-ornithine.     

The genetic incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast

The noncanonical amino acid p-azidomethyl-l-phenylalanine can be genetically incorporated into proteins in bacteria, and has been used both as a spectroscopic probe and for the selective modification of proteins by alkynes using click chemistry. Here we report identification of Escherichia coli tyrosyl tRNA synthetase mutants that allow incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast. When expressed together with the cognate E. coli tRNA_CUA^Tyr, the new mutant tyrosyl tRNA synthetases directed robust incorporation of p-azidomethyl-l-phenylalanine into a model protein, human superoxide dismutase, in response to the UAG amber nonsense codon. Mass spectrometry analysis of purified superoxide dismutase proteins confirmed the efficient site-specific incorporation of p-azidomethyl-l-phenylalanine. This work provides an additional tool for the selective modification of proteins in eukaryotic cells.     

Single crystals of a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine

Lamellar single crystals were formed from a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine at the ratio of 4 to 1. The copolypeptide takes the αhelical structure. The crystals were formed by casting dilute solutions at room temperature from a solvent consisting of a 1 to 1 mixture of chloroform and trifluoroacetic acid and were observed by electron microscopy. The average crystal thickness was 670 a in the as-polymerized sample, and 580 a in a fractionated sample. The thickness was decreased by annealing at temperatures above 110 C. A hexagonal form, a group of three orthorhombic forms (group 1), and a group of an orthorhombic form and two monoclinic forms (group II) were observed by electron diffraction. The diversity of the crystal structures is suggested to be caused by a variation in crystallization conditions during evaporation of the solvent. The hexagonal form and the structures of group I are changed into the structures of group II by annealing. The crystal structures other than the hexagonal form indicate on ordered arrangements of side chains in the crystals.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Metabolism of 14C-labelled D-glucose, D-fructose and allitol by Itea plants

The metabolism of D-[1-¹⁴C]glucose, D-[6-¹⁴C]glucose, D-[1-¹⁴C]fructose and D-[6-¹⁴C]fructose by leafy spurs of Itea plants results in rapid incorporation of label into allitol and D-allulose. The patterns of labelling found in the allitol and D-allulose are discussed, a direct interconversion from D-glucose and D-fructose being indicated. Allitol has been found to be an active metabolite in Itea plants.     

Subunit composition and substrate binding region of potato l-lactate dehydrogenase

Four of the six electrophoretically distinguishable isoenzymes of the l-lactate dehydrogenase (EC 1.1.1.27) from potato tubers were purified from crude extracts. The isoenzymes are tetrameric and exhibit MWs around 145000. They are composed of mixtures of different subunits. Two of the isoenzymes together contain at least three, the other two together contain six different subunits indicating that the actual number of isoenzymes may be even greater than the number of electrophoretically detectable isoenzymes. Since the isoenzymes agree largely with respect to their enzymatic properties and to their primary structure as suggested from fingerprinting and amino acid analysis, it is suggested that the variation of the subunits is caused by proteolytic processing in vivo rather than by different genetic coding. The amino acid sequence of the substrate-binding region (Arg₆ peptide) shows a high homology to that of the l-lactate dehydrogenases of animals and bacteria indicating a common origin of plant, animal and bacterial enzymes.     

Partial purification and characterization of a ribonuclease from Petunia hybrida

A crude extract from leaves of Petunia hybrida contains three ribonucleases (RNases) as revealed by polyacrylamide gel electrophoresis (PAGE). One     

Synthesis of aminocyclitols from l-quinic acid

A variety of 1,3-diamino and 1,4-diaminocyclitols, manoaminocyclitols, and triaminocyclohexanol have been synthesized starting with the chiral ketone intermediate, 2, derived from l-quinic acid. Reduction of 2 with lithium borohydride afforded two epimeric diols (4 and 5), both of which were transformed by straight-forward but distinctly different chemical procedures into potentially useful aglycons for preparing novel tupes of bioactive, aminocyclitol glycoside antibiotics. The disposition of the substituents at C-1, C-3, C-4, and C-5 in 19 and 37 is identical with that present in the 2-deoxystreptamine nucleus in the naturally occurring antibiotics     

Detection and properties of l-histidinol dehydrogenase in wheat germ

The presence and partial characterization of the properties of l-histidinol dehydrogenase (EC 1.1.1.23), the enzyme catalysing the last step in the pathway of histidine biosynthesis, has been described in higher plants for the first time. The activity has been found in cell-free extracts from wheat germ, turnip root, radish root and squash fruit. The enzyme has been partially purified and characterized from extracts of acetone powders of wheat germ. DEAE-cellulose chromatography revealed two peaks of histidinol dehydrogenase activity. In one there was a rapid reduction of NAD⁺ in the absence of histidinol; however, the rate was stimulated by the addition of histidinol. The rate in the absence of substrate became quite low after several min and the histidinol-dependent rate was then easily observed. The second peak of activity did not reduce NAD⁺ unless l-histidinol was present in the assay mixture. The K^ms for l-histidinol and NAD⁺ were determined for this latter enzyme. The values obtained at saturating concentrations of the other substrate were l-histidinol, 8.8 μM and NAD⁺, 0.14 mM. The product of the dehydrogenase reaction was histidine as determined by paper chromatography.     

Phenylalanine ammonia-lyase: A model for the cooperativity kinetics induced by d- and l-phenylalanine

A preparation of l-phenylalanine ammonia-lyase (EC 4.1.3.5.) from soybean (Glycine max L. cv. Kanrich) showed negative cooperativity with respect to l-phenylalanine and competitive inhibition by d-phenylalanine. A two-protomer partially concerted model for inhibition kinetics is described. If cooperativity is associated with ligand binding but not k_cat, plots of v against log [S] at constant [I] are symmetrical. Such curves may be fitted by graphical or iterative least-squares methods. The experimental results conform to this restricted model. The three-substrate and three-inhibitor dissociation constants were estimated by a stepwise procedure. For substrate only the first and second dissociation constants were 12 and 78 μm, respectively, with a symmetry point value of 30.5 μm. To a first approximation, site occupancy determines the cooperativity. As d- and l-phenylalanine produce equivalent effects, they are assumed to pack into the same induced space. As ligand binding at one site has little influence on the relative d:l binding at the other and does not influence k_cat, cooperativity probably reflects changes in regions remote from the active site such as the interface between the protomers. The regulatory range in [S] of the enzyme in vivo may be indicated by the linearity range of the semilog plot for the isolated enzyme. The observed range corresponds to a 100-fold change in [S] compared to a 10-fold change for Michaelis-Menten kinetics.     

γ-l-Glutamyl-l-pipecolic acid in Gleditsia caspica

y-l-Glutamyl-l-pipecolic acid has been isolated from seeds of Gleditsia caspica (L.) Desf. Proof of its structure was obtained by chromatographic and spectroscopic examination of the natural product and its hydrolytic products. The new compound is the first example of a naturally occurring γ-glutamyl imino acid.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

Cinnamate and shikimate incorporation into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins: Are there alternative pathways?

The incorporation of shikimate-[¹⁴C] and cinnamate-[¹⁴C] into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins was studied in isolated petals of Petunia hybrida. According to the dilution values, the incorporation of shikimate-[¹⁴C] was about 3–6 times better than that of cinnamate-[¹⁴C]. However a comparison of the incorporation of the 2 precursors on a relative basis showed no significant differences in the relative proportions of the specific activities of the 3′,4′-dihydroxysubstituted cyanidin-3-monoglucoside and the 3′,4′,5′-trihydroxysubstituted delphinidin-3-monoglucoside. This result and the [¹⁴C]-incorporation behaviour of the 3′-methoxy-4′-hydroxysubstituted peonidin-3-monoglucoside do not support the hypothesis that there are alternative pathways of flavonoid biosynthesis.