Can the combination of electrochemical regeneration of NAD+, selectivity of L‐α‐amino‐acid dehydrogenase,and reductive amination of α‐keto‐acid be applied to the inversion of configuration of a L‐α‐amino‐acid?

The inversion of configuration of L‐alanine can be carried out by combining its selective oxidation in the presence of NAD⁺ and L‐alanine dehydrogenase, electrochemical regeneration of the NAD⁺ at a carbon felt anode, and reductive amination of pyruvate, i.e., reduction of its imino derivative at a mercury cathode, the reaction mixture being buffered with concentrated ammonium/ammonia (1.28M / 1.28M). The dehydrogenase exhibits astonishing activity and stability under such extreme conditions of pH and ionic strength. The main drawback of the process is its slowness. At best, the complete inversion of a 10 mM solution of L‐alanine requires 140 h. A careful and detailed quantitative analysis of each of the key steps involved shows that the enzyme catalyzed oxidation is so thermodynamically uphill that it can be driven efficiently to completion only when both the coenzyme regeneration and the pyruvate reduction are very effective. The first condition is easily fulfilled. Under the best conditions, it is the rate of the chemical reaction producing the imine which controls the whole process kinetically. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 101–107, 1999.     

Fatty acid ω-hydroxylases from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1.

Abstract

Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

     

Simultaneous determination and quantification of three pentacyclic triterpenoids—betulinic acid,oleanolic acid,and ursolic acid—in cell cultures of <Emphasis Type="BoldItalic">Lantana camara</Emphasis> L.

A simple procedure has been described for simultaneous determination and improved yield of three pentacyclic triterpenoids—betulinic, oleanolic, and ursolic acids—from callus cultures of Lantana camara. Cell biomass was obtained from leaf disk explants cultured on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 5 μM 6-benzylaminopurine, 1 μM 2,4-dichlorophenoxyacetic acid, and 1 μM α-naphthaleneacetic acid. Optimum separation of the three compounds was achieved by reverse-phase high-pressure liquid chromatography on a C₁₈ column with 80:20 (v/v) acetonitrile/water as mobile phase. With this route, a yield of 3.1% betulinic acid, 1.88% oleanolic acid, and 4.12% ursolic acid per gram dry weight was obtained from cultures. Leaves from the parent plant, used as control, showed total absence of betulinic acid, and the quantities of oleanolic and ursolic acids present in them were only marginally higher than that found in in vitro-raised cultures. Presence of the three compounds was further confirmed by electrospray ionization mass spectrometry.     

Level of accumulation of epoxy fatty acid in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> expressing a linoleic acid Δ12-epoxygenase is influenced by the availability of the substrate linoleic acid

Arabidopsis thaliana (L.) Heynh. expressing the Crepis palaestina (L.) linoleic acid 12-epoxygenase in its developing seeds typically accumulates low levels of vernolic acid (12,13-epoxy-octadec-cis-9-enoic acid) in comparison to levels found in seeds of the native C. palaestina. In order to determine some of the factors limiting the accumulation of this unusual fatty acid, we have examined the effects of increasing the availability of linoleic acid (9cis, 12cis-octadecadienoic acid), the substrate of the 12-epoxygenase, on the quantity of epoxy fatty acids accumulating in transgenic A. thaliana. The addition of linoleic acid to liquid cultures of transgenic plants expressing the 12-epoxygenase under the control of the cauliflower mosaic virus 35S promoter increased the amount of vernolic acid in vegetative tissues by 2.8-fold. In contrast, the addition to these cultures of linoelaidic acid (9trans, 12trans-octadecadienoic acid), which is not a substrate of the 12-epoxygenase, resulted in a slight decrease in vernolic acid accumulation. Expression of the 12-epoxygenase under the control of the napin promoter in the A. thaliana triple mutant fad3/fad7-1/fad8, which is deficient in the synthesis of tri-unsaturated fatty acids and has a 60% higher level of linoleic acid than the wild type, was found to increase the average vernolic acid content of the seeds by 55% compared to the expression of the 12-epoxygenase in a wild-type background. Together, these results reveal that the availability of linoleic acid is an important factor affecting the synthesis of epoxy fatty acid in transgenic plants.     

Do electrostatic interactions destabilize protein–nucleic acid binding?

The negatively charged phosphates of nucleic acids are often paired with positively charged residues upon binding proteins. It was thus counter-intuitive when previous Poisson-Boltzmann (PB) calculations gave positive energies from electrostatic interactions, meaning that they destabilize protein-nucleic acid binding. Our own PB calculations on protein-protein binding have shown that the sign and the magnitude of the electrostatic component are sensitive to the specification of the dielectric boundary in PB calculations. A popular choice for the boundary between the solute low dielectric and the solvent high dielectric is the molecular surface; an alternative is the van der Waals (vdW) surface. In line with results for protein-protein binding, in this article, we found that PB calculations with the molecular surface gave positive electrostatic interaction energies for two protein-RNA complexes, but the signs are reversed when the vdW surface was used. Therefore, whether destabilizing or stabilizing effects are predicted depends on the choice of the dielectric boundary. The two calculation protocols, however, yielded similar salt effects on the binding affinity. Effects of charge mutations differentiated the two calculation protocols; PB calculations with the vdW surface had smaller deviations overall from experimental data.     

The decomposition of α-aminophosphine oxides to phosphonic acid derivatives (P<Superscript>III</Superscript>)

Aminophosphine oxides and aminophosphonates are, in general, very stable compounds. However, following phosphorus–carbon bond cleavage in aqueous acidic media these compounds sometimes decompose to phosphonic acids derivatives (P^III). Despite some controversy in the literature, careful analysis supported by theoretical studies leads to the conclusion that decomposition to P^III derivatives proceeds via an elimination reaction. Figure The decomposition of α-aminophosphine oxides to phosphonic acid derivatives (P^III)     

Fatty acid composition and <Emphasis Type="Italic">PlFADs</Emphasis> expression related to α-linolenic acid biosynthesis in herbaceous peony (<Emphasis Type="Italic">Paeonia lactiflora</Emphasis> Pall.)

As a new α-linolenic acid (ALA) resource, there has been little known about the relationship between expression levels of fatty acid desaturase (FAD) genes during seed development and fatty acid (FA) composition in herbaceous peony (Paeonia lactiflora Pall.). In this study, oil content and FA composition of nine cultivars were measured at four different stages during seed development. Moreover, five genes including PlFAD2-1, PlFAD2-2, PlFAD3-2, PlFAD6 and PlFAD7 related to the ALA biosynthesis were isolated. Furthermore, the relative expression levels of these genes in seeds were investigated in two cultivars, that is, ‘PL1’ and ‘PL2’ with higher and lower ALA content, respectively. The results showed that oil content was from 17.03 ± 0.15 to 24.51 ± 0.15%; 15 kinds of FA were detected. However, the relative content of ALA was decreased during seed development. Although, the genes PlFAD2-1, PlFAD2-2, PlFAD3-2, PlFAD6 and PlFAD7 were all expressed during the seed development, the expression levels varied significantly. Most of the genes were expressed strongly in the early stage but weakly in the late stage except PlFAD6. Moreover, expression level of gene PlFAD2-2 was the highest while that of PlFAD7 was lowest at S1. In addition, the relative expression level of genes in ‘PL1’ (high ALA content) was higher than those in ‘PL2’ (low ALA content) during the early stage of the seed development apart from gene PlFAD6. This study provides a reliable theoretical basis for improving the contents of ALA by means of genetic engineering in the herbaceous peony seed oil.     

δ-Aminolaevulinic acid and amino acid neurotransmitters

Summary The effects of the porphyrin precursor -aminolaevulinic acid (ALA) on -aminobutyric acid (GABA) and L-glutamate transmitter systems was investigated in rat brain. It was found that ALA inhibited GABA and glutamate uptake and stimulated basal efflux of the amino acids in purified nerve endings. These effects were evident only at relatively high concentrations of ALA (at least 100 M). Such concentrations probably do not occur in the nervous systems of patients suffering from acute porphyria. In addition, it was found that ALA inhibited the stimulated release of GABA from nerve endings probably by acting as an agonist at GABA autoreceptors. This effect was found at very low concentrations of ALA (1 M). It is therefore likely that the neuropsychiatric manifestations of the acute porphyric attack are attributable, to some extent, to reduced GABA release at central synapses.     

<Emphasis Type="Italic">γ</Emphasis>-Fluorinated analogues of glutamic acid and glutamine

Gamma-fluorinated analogues of glutamic acid and glutamine are compounds of biological interest. Syntheses of such compounds are extensively reviewed in this article. 4-fluoroglutamic acid was prepared as a mixture of racemic diastereomers by Michael reaction, inverse-Michael reaction or by electrophilic / nucleophilic fluorination. Optically enriched 4-fluoroglutamic acids were obtained by several resolution techniques as well as by asymmetric methodologies using the chiral pool. 4-fluoroglutamine was prepared as a mixture of stereoisomers as well as in racemic erythro and threo forms from the corresponding 4-fluoroglutamic acids using aminolysis and conventional protection and deprotection strategies. Racemic 4,4-difluoroglutamic acid was synthesized by a nitroaldol reaction and its L-enantiomer obtained via three different asymmetric routes. Racemic 4,4-difluoroglutamic acid was converted into the corresponding 4,4-difluoroglutamine using a protection / aminolysis / deprotection sequence while N-Boc-L-4,4-difluoroglutamine was prepared directly from (R)-Garner's aldehyde using a Reformatsky reaction as the key step.     

Photosynthetic pigments content, <Emphasis Type="Italic">δ</Emphasis>-aminolevulinic acid dehydratase and acid phosphatase activities and mineral nutrients concentration in cadmium-exposed <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

In this study, the effects of cadmium chloride (CdCl₂) on plant growth, histology of roots, photosynthetic pigments content, δ-aminolevulinic acid dehydratase (ALA-D; E.C. 4.2.1.24) and acid phosphatase activities (AP; E.C. 3.1.3.2), soluble phosphorus (Pi) measurement and mineral nutrients content in cucumber seedlings (Cucumis sativus L.) were investigated. Cucumber seedlings were grown in vitro in an agar-solidified substrate containing four CdCl₂ treatments (0, 100, 400, and 1000 μM) for ten days. Cd was readily absorbed by seedlings and its content was greater in the roots than in the shoot. Cd reduced shoot and root length, and fresh and dry biomass of seedlings. Inhibition of root cell elongation in Cd-treated seedlings was observed by the increase of the mean radial size of cells belonging to three zones of the root tip. The highest level of Cd reduced in a similar manner chlorophyll a, chlorophyll b and total chlorophyll contents. Increasing concentrations of Cd resulted in a linear decrease in carotenoids levels of cotyledons. Interestingly, the ALA-D activity in cotyledons was inhibited only at the highest level of Cd. Root and shoot AP activities were, respectively, activated and inhibited at all CdCl₂ concentrations. Root Pi concentration was increased in all Cd treatments and it was not altered in the shoot tissues. Moreover, in general, the nutrient contents were increased in the root and decreased in the shoot. Therefore, we suggest that Cd affects negatively growth, photosynthetic pigments, ALA-D and AP activities and partition of mineral nutrients in cucumber seedlings.     

Secretion of acid phosphatase in<Emphasis Type="Italic">Claviceps purpurea</Emphasis> — an ultracytochemical study

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.     

Synthesis of C<Subscript>3</Subscript>-symmetric and C<Subscript>4</Subscript>-symmetric amino acid derivatives via Suzuki–Miyaura cross-coupling reaction

Various non-natural C(3)- and C(4)-symmetric alpha-amino acid derivatives have been synthesized via Suzuki-Miyaura cross-coupling reaction between aromatic iodides or bromide and a suitably protected DL-4-boronophenylalanine derivative.     

Glutamic acid – amino acid,neurotransmitter, and drug – is responsible for protein synthesis rhythm in hepatocyte populations <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell–cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.     

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in <Emphasis Type="Italic">Mentha</Emphasis> × <Emphasis Type="Italic">piperita</Emphasis> cell suspension cultures

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g⁻¹ DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g⁻¹ DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.     

Do stress-related phytohormones,abscisic acid and jasmonic acid play a role in the regulation of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. somatic embryogenesis?

This study examined the role of endogenous abscisic acid (ABA) and jasmonic acid (JA) in indirect somatic embryogenesis of Medicago sativa L. A multiplex GC-MS/MS technique allowed quantitative single-run analyses of ABA, JA, 12-oxophytodienoic acid (OPDA) and indole-3-acetic acid (IAA). The preparation of initial explants led to a strong accumulation of ABA, JA and OPDA but not of IAA. Substantially higher levels of ABA, JA and OPDA were detected in developing somatic embryos than in callus or embryogenic suspension. Fluridone (FLD) decreased ABA, JA and OPDA levels. Indoprofen (INP) appeared to be a specific inhibitor of octadecanoid biosynthesis. Somatic embryo production and development were negatively affected by FLD or INP. Only INP (0.5 μM) applied during proliferation phase increased the number of cotyledonary embryos. The results strongly indicate the involvement of ABA and JA in somatic embryogenesis of M. sativa. Surprisingly, low IAA contents in comparison to stress-related compounds (ABA, JA and OPDA) were detected in explants, embryogenic tissues and somatic embryos.     

Synthesis of α-cyclopiazonic acid by fungi of the genus <Emphasis Type="Italic">Aspergillus</Emphasis>

The presence of α-cyclopiazonic acid has been studied among metabolites of Aspergillus fungi. The study was performed with 138 cultures of 13 species obtained from the All-Russia Collection of Microorganisms and the collection of our institute. α-Cyclopiazonic acid was most frequently encountered among the metabolites of the section Flavi (the ability to synthesize α-cyclopiazonic acid was expressed in 61% of the strains of A. flavus, 83% of the strains of A. oryzae, and all strains of A. tamarii). This expression index for A. versicolor was less than 5%. We showed for the first time that α-cyclopiazonic acid is produced by A. fumigatus and A. phoenicis (expression in 30% of the strains of either species).     

Metabolic flux distribution for γ-linolenic acid synthetic pathways in<Emphasis Type="Italic">Spirulina platensis</Emphasis>

Spirulina produces γ-linolenic acid (GLA), an important pharmaceutical substance, in a relatively low level compared with fungi and plants, prompting more research to improve its GLA yield. In this study, metabolic flux analysis was applied to determine the cellular metabolic flux distributions in the GLA synthetic pathways of twoSpirulina strains, wild type BP and a high-GLA producing mutant Z19/2. Simplified pathways involving the GLA synthesis ofS. platensis formulated comprise of photosynthesis, gluconeogenesis, the pentose phosphate pathway, the anaplerotic pathway, the tricarboxylic cycle, the GLA synthesis pathway, and the biomass synthesis pathway. A stoichiometric model reflecting these pathways contains 17 intermediates and 22 reactions. Three fluxes—the bicarbonate (C-source) uptake rate, the specific growth rate, and the GLA synthesis rate—were measured and the remaining fluxes were calculated using linear optimization. The calculation showed that the flux through the reaction converting acetyl-CoA into malonyl-CoA in the mutant strain was nearly three times higher than that in the wild-type strain. This finding implies that this reaction is rate controlling. This suggestion was supported by experiments, in which the stimulating factors for this reaction (NADPH and MgCl₂) were added into the culture medium, resulting in an increased GLA-synthesis rate in the wild type strain.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.