First description of betalains biosynthesis in an aquatic organism: characterization of 4,5-DOPA-extradiol-dioxygenase activity in the cyanobacteria Anabaena cylindrica

The biosynthesis of betalamic acid, the structural unit of pigments betalains, is performed by enzymes with 4,5-DOPA-extradiol-dioxygenase activity. These enzymes were believed to be limited to plants of the order Caryophyllales and to some fungi. However, the discovery of Gluconacetobacter diazotrophicus as the first betalain-forming bacterium opened a new field in the search for novel biological systems able to produce betalains. This paper describes molecular and functional characterization of a novel dioxygenase enzyme from the aquatic cyanobacterium Anabaena cylindrica. The enzyme was found to be a homodimer of a polypeptide of 17.8 kDa that, opposite to previous related enzymes, showed a strong inhibition by excess of the precursor L-DOPA. However, its heterologous expression has allowed detecting the formation of the main compounds in the biosynthetic pathway of betalains. In addition, phylogenetic analysis has shown that this enzyme is not close related to enzymes from plants, fungi or proteobacteria such as G. diazotrophicus. The presence of enzymes that produce these health-promoting compounds is more diverse than expected. The discovery of this novel dioxygenase in the phylum cyanobacteria expands the presence of betalamic acid-forming enzymes in organisms of different nature with no apparent relationship among them.     

A dopamine-based biosynthetic pathway produces decarboxylated betalains in Chenopodium quinoa

Betalains are the nitrogenous pigments that replace anthocyanins in the plant order Caryophyllales. Here, we describe unconventional decarboxylated betalains in quinoa (Chenopodium quinoa) grains. Decarboxylated betalains are derived from a previously unconsidered activity of the 4,5-DOPA-extradiol-dioxygenase enzyme (DODA), which has been identified as the key enzymatic step in the established biosynthetic pathway of betalains. Here, dopamine is fully characterized as an alternative substrate of the DODA enzyme able to yield an intermediate and structural unit of plant pigments: 6-decarboxy-betalamic acid, which is proposed and described. To characterize this activity, quinoa grains of different colors were analyzed in depth by chromatography, time-of-flight mass spectrometry, and reactions were performed in enzymatic assays and bioreactors. The enzymatic-chemical scheme proposed leads to an uncharacterized family of 6-decarboxylated betalains produced by a hitherto unknown enzymatic activity. All intermediate compounds as well as the final products of the dopamine-based biosynthetic pathway of pigments have been unambiguously determined and the reactions have been characterized from the enzymatic and functional perspectives. Results evidence a palette of molecules in quinoa grains of physiological relevance and which explain minor betalains described in plants of the Caryophyllales order. An entire family of betalains is anticipated.

A biosynthetic pathway produces unconventional plant pigments betalains derived from dopamine in quinoa.     

Scaled-up biotechnological production of individual betalains in a microbial system

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.     

Biogenesis of betalamic acid

When d,l-dopa-[1-¹⁴C] and -[2-¹⁴C] was fed to yellow flower buds of Portulaca grandiflora betalamic-[¹⁴C] acid was obtained. The labeled betalamic acid was converted to ¹⁴C-labeled betanin in order to obtain a stable substance which could be recrystallized to a radio-pure sample. Decarboxylation of the radiopure betanin obtained from the sequence using dopa-[1-¹⁴C] indicated that the ¹⁴C-carboxyl group of dopa corresponded to a ¹⁴C-carboxyl group in betanin and hence in betalamic acid. The shape of the ORD curve for the naturally occurring betalamic acid was the same as that recorded for a sample of [S]-betalamic acid derived by degradation of betanin. These data support the hypothesis that betalamic acid is formed in vivo by an oxidative cleavage of l-dopa and that it is an intermediate in the biogenesis of other betalains from dopa.     

Structural implications on color, fluorescence, and antiradical activity in betalains

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors on flowers and fruits of most plants of the order Caryophyllales. They are classified as betacyanins, exhibiting a violet coloration, and betaxanthins, which exhibit yellow coloration. Traditionally, betalains have been defined as condensation products of betalamic acid with different amines and amino acids, but the implication of the pigment structure for their properties has not been investigated. This paper explores different structural features of the betalains, revealing the clues for the switch from yellow to violet color, and the loss of fluorescence. A relevant series of 15 betalain-related compounds (both natural and novel semisynthetic ones) is obtained and characterized by chromatography, UV-vis spectrophotometry, fluorescence, and electrospray ionization mass spectroscopy. Antiradical properties of individual pure compounds in a broad pH range are studied under the ABTS^•+ radical assay. Relevance of specific bonds is studied, and differences between betaxanthins and betacyanins are used to explore in depth the structure–antiradical activity relationships in betalains.     

Characterization and functional identification of a novel plant 4,5-extradiol dioxygenase involved in betalain pigment biosynthesis in Portulaca grandiflora

Betalains are pigments that replace anthocyanins in the majority of families of the plant order Caryophyllales. Betalamic acid is the common chromophore of betalains. The key enzyme of the betalain biosynthetic pathway is an extradiol dioxygenase that opens the cyclic ring of dihydroxy-phenylalanine (DOPA) between carbons 4 and 5, thus producing an unstable seco-DOPA that rearranges nonenzymatically to betalamic acid. A gene for a 4,5-DOPA-dioxygenase has already been isolated from the fungus Amanita muscaria, but no homolog was ever found in plants. To identify the plant gene, we constructed subtractive libraries between different colored phenotypes of isogenic lines of Portulaca grandiflora (Portulacaceae) and between different stages of flower bud formation. Using in silico analysis of differentially expressed cDNAs, we identified a candidate showing strong homology at the level of translated protein with the LigB domain present in several bacterial extradiol 4,5-dioxygenases. The gene was expressed only in colored flower petals. The function of this gene in the betalain biosynthetic pathway was confirmed by biolistic genetic complementation in white petals of P. grandiflora genotypes lacking the gene for color formation. This gene named DODA is the first characterized member of a novel family of plant dioxygenases phylogenetically distinct from Amanita sp. DOPA-dioxygenase. Homologs of DODA are present not only in betalain-producing plants but also, albeit with some changes near the catalytic site, in other angiosperms and in the bryophyte Physcomitrella patens. These homologs are part of a novel conserved plant gene family probably involved in aromatic compound metabolism.     

Betalains in the era of global agri-food science, technology and nutritional health

Natural pigments from plants are of growing interest as substitutes for synthetic dyes in the food and pharmaceutical industry and they increase their added value if they possess positive effects on health. These pigments can be added as such if they are in the legal authorized lists of additives or can be added as phytochemical-enriched plant extract achieving the original product, which has received it, the new nomenclature of functional food. In this way, we comprise on this review a wide point of view of a group of natural pigments known as betalains. From a chemical point of view, betalains are ammonium conjugates of betalamic acid with cyclo-DOPA (betacyanins, violet) and aminoacids or amines (betaxanthins, orange or yellow), which are compounds present in our diet. Besides and taking into account that one type of betalain, betanin is approved as food colorant (E-162) by the European Union and that enlarges the specific weight of these compounds in the diet, we have evolved an overview from the biosynthesis, technology and promoting production, industrial uses as pigments up to physiological and nutritional biovailability or biological and health-promoting properties of betalains for accessible information to industrials, researchers and consumers.     

Betalains of Celosia argentea

The betalains of yellow, orange and red inflorescences of common cockscomb (Celosia argentea var. cristata) were compared and proved to be qualitatively identical to those of feathered amaranth (Celosia argentea var. plumosa). In addition to the known compounds amaranthin and betalamic acid, the structures of three yellow pigments were elucidated to be immonium conjugates of betalamic acid with dopamine, 3-methoxytyramine and (S)-tryptophan by various spectroscopic techniques and comparison to synthesized reference compounds; the latter two are new to plants. Among the betacyanins occurring in yellow inflorescences in trace amounts, the presence of 2-descarboxy-betanidin, a dopamine-derived betacyanin, has been ascertained. The detection of high dopamine concentration may be of toxicological relevance in use of yellow inflorescences as a vegetable and in traditional Chinese medicine, common uses for the red inflorescences of common cockscomb.     

Betacyanin decolourizing enzyme in Amaranthus tricolor seedlings

The characterization and partial purification of an enzyme from Amaranthus tricolor which decolourizes betacyanin are described. The enzyme occurs in greatest amounts in the roots and in 3.5-4-day-old seedings. Preparation from an acetone powder of roots results in a more active and more stable enzyme than that obtained from crude buffer extraction. The activity is in the 130 000 g supernatant from sucrose-buffer extraction. It has a pH optimum of 3.4 K_m towards amaranthin of 3.1 × 10^?6 M and towards betanin of 3.5 × 10^?6 M, and is inhibited by lack of oxygen, and by azide, diethyldithiocarbamate, thiourea, dithiothreitol and cysteine. The product of the reaction has the spectral and electrophoretic properties of betalamic acid. The possibility of enzymic decolourization of betacyanin during acetic acid extraction used for assay of the pigment in the Amaranthus bioassay for cytokinins needs to be recognized.     

Humilixanthin a new betaxanthin from Rivina humilis

A new betalain has been isolated from fruits ofRivina humilis and identified as the betaxanthin humilixanthin, the 5-hydroxynorvaline-immonium conjugate of betalamic acid. Its structure was elucidated and characterized by¹H NMR spectroscopy, FAB mass and GC/MS spectrometry, UV/Vis absorption spectroscopy, high-performance liquid chromatography, thin-layer chromatography and electrophoresis. The structure of the amino acid moiety 5-hydroxynorvaline (2-amino-5-hydroxyvaleric acid) was unambiguously confirmed by comparison with synthetic reference material. Humilixanthin was also detected in fruits ofPhytolacca acinosa andP. bogotensis, in petals ofDelosperma luteum,Lampranthus aurantiacus,L. peersii,Portulaca grandiflora, and in the yellow-coloured root ofBeta vulgaris.     

Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile

A nitrilase that converts racemic mandelonitrile to R-(—)-mandelic acid was purified to apparent homogeneity from a cell extract of Alcaligenes faecalis ATCC 8750. The molecular weight of this enzyme was estimated to be 32,000±2,000 from SDS-PAGE and that of the native enzyme 460,000±30,000 from HPLC gel filtration. The enzyme preferentially hydrolyzed substituted aliphatic nitriles, in particular benzyl cyanide and its p-substituted compounds, but hydrolyzed aromatic nitriles only with difficulty. The amino-terminal amino acids were sequenced and their sequences compared with those of other nitrilases. The purified enzyme had a pH optimum of 7.5 and an optimum temperature range of 40 to 45°C. The enzyme was inhibited by various thiol reagents. It hydrolyzed racemic mandelonitrile, producing optically pure R-(—)-mandelic acid and ammonia without the concomitant production of mandelamide, evidence that this nitrilase is highly enantioselective for R-mandelonitrile.     

The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris “Golden Beet”) showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris “Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.     

Biochemical complementation of the betalain biosynthetic pathway in Portulaca grandiflora by a fungal 3,4-dihydroxyphenylalanine dioxygenase

3,4-Dihydroxyphenylalanine (DOPA) dioxygenase from Amanitamuscaria catalyses the key reaction of betalain biosynthesis, namely the conversion of DOPA to betalamic acid by a 4,5-ring-opening reaction. In addition, it catalyses a 2,3 opening which yields the fungal pigment muscaflavin, a compound that has never been found in plants. In this work, a cDNA clone (DodA) encoding A. muscaria DOPA-dioxygenase was expressed in white Portulacagrandiflora petals, using the particle bombardment technique. Transformation resulted in the formation of yellow and violet spots that contained betalain pigments and muscaflavin, indicating that the fungal enzyme was expressed and active in plants, and could complement the plant betalain biosynthetic pathway. The presence of muscaflavin in transformed plants indicates a difference in the specificity of the plant and A.muscaria enzymes. Received: 24 February 1997 / Accepted: 20 May 1997     

Characterization of 12-oxo-phytodienoic Acid reductase in corn: the jasmonic Acid pathway

12-Oxo-phytodienoic acid reductase, an enzyme of the biosynthetic pathway that converts linolenic acid to jasmonic acid, has been characterized from the kernel and seedlings of corn (Zea mays L.). The molecular weight of the enzyme, estimated by gel filtration, was 54,000. Optimum enzyme activity was observed over a broad pH range, from pH 6.8 to 9.0. The enzyme had a K_m of 190 micromolar for its substrate, 12-oxo-phytodienoic acid. The preferred reductant was NADPH, for which the enzyme exhibited a K_m of 13 micromolar, compared with 4.2 millimolar for NADH. Reductase activity was low in the corn kernel but increased five-fold by the fifth day after germination and then gradually declined.     

Crystal structure of glutamate-1-semialdehyde aminotransferase from Bacillus subtilis with bound pyridoxamine-5'-phosphate

Glutamate-1-semialdehyde aminotransferase (GSA-AT), also named glutamate-1-semialdehyde aminomutase (GSAM), a pyridoxamine-5′-phosphate (PMP)/pyridoxal-5′-phosphate (PLP) dependent enzyme, catalyses the transamination of the substrate glutamate-1-semialdehyde (GSA) to the product 5-Aminolevulinic acid (ALA) by an unusual intramolecular exchange of amino and oxo groups within the catalytic intermediate 4,5-diaminovalerate (DAVA). This paper presents the crystal structure of GSA-AT from Bacillus subtilis (GSA-AT_Bsu) in its PMP-bound form at 2.3 Å resolution. The structure was determined by molecular replacement using the Synechococcus GSAM (GSAM_Syn) structure as a search model. Unlike the previous reported GSAM/GSA-AT structures, GSA-AT_Bsu is a symmetric homodimer in the PMP-bound form, which shows the structural symmetry at the gating loop region with open state, as well as identical cofactor (PMP) binding in each monomer. This observation of PMP in combination with an “open” lid supports one characteristic feature for this enzyme, as the catalyzed reaction is believed to be initiated by PMP. Furthermore, the symmetry of GSA-AT_Bsu structure challenges the previously proposed negative cooperativity between monomers of this enzyme.     

Establishment and characterization of a betaxanthin-producing cell culture from Portulaca grandiflora

Cell cultures derived from three yellow flowering Portulaca grandiflora genotypes contained betacyanins rather than betaxanthins. A betaxanthin-producing cell culture was obtained by subculturing orange cell clusters isolated from the red-violet cell culture of a violet flowering P. grandiflora genotype. Selection of the most strongly yellow coloured cell material reduced the portion of betacyanins considerably and resulted in a P. grandiflora cell culture characterized by a high concentration of betaxanthins and the occurrence of free betalamic acid. Vulgaxanthin I was the main compound. Besides these pigments carotenoids and flavonoids were detectable.Betaxanthin biosynthesis strongly dependend on light. Product accumulation reached its maximum during the stationary phase of the growth cycle. Excretion of pigments, especially of betalains, could not be detected. Vulgaxanthin I as found in the cell culture was identical with one of two main betaxanthins in the yellow petals of P. grandiflora.The yellow P. grandiflora cells grew well on solid modified Murashige and Skoog medium but failed to grow in liquid medium after a few subcultures. In contrast, white P. grandiflora suspension cultures could be established repeatedly.Abbreviations A absorbance - BX I and BX II betaxanthin fractions - DOPA dihydroxyphenylalanin - DW cell dry weight - molar extinction coefficient - SS solvent system     

Application of fast atom bombardment mass spectrometry to chlorogenic acids

The technique of positive- and negative-ion fast atom bombardment mass spectrometry has been shown to be capable of producing molecular mass and useful fragmentation information for the structural elucidation of chlorogenic acids. The mass spectra of chlorogenic acid and the related compounds 3′-O-methylchlorogenic acid, neochlorogenic acid, 4,5-dicaffeoyl quinic acid and 1,5-dicaffeoyl quinic acid are compared with those obtained by electron impact mass spectrometry.     

Molecular and catalytic properties of the acetoacetyl-coenzyme A thiolase of Escherichia coli.

The acetoacetyl-CoA-thiolase, a product of the acetoacetate degradation operon (ato) was purified to homogeneity as judged by polyacrylamide-gel electrophoresis at pH 4.5, 7.0, and 8.3. The enzyme has a molecular weight of 166,000 and is composed of four identical subunits. The subunit molecular weight is 41,500. Histidine was the sole N-terminal amino acid detected by dansylation. The thiolase contains eight free sulhydryl residues and four intrachain disulfide bonds per mole. The ato thiolase catalyzes the CoA- dependent cleavage of acetoacetyl-CoA and the acetylation of acetyl-CoA to form acetoacetyl-CoA. The maximal velocity in the direction of acetoacetyl-CoA cleavage was 840 nmol min^? (enzyme unit)^?1 and the maximal velocity in the direction of acetoacetyl CoA formation was 38 nmol min^?1 (enzyme unit)^?1. Like other thiolases, the ato thiolase was inactivated by sulfhydryl reagents. The enzyme was protected from inactivation by sulfhydryl reagents in the presence of the acyl-CoA substrates, acetyl-CoA and acetoacetyl-CoA; however, no protection was obtained when the enzyme was incubated with the acetyl-CoA analog, acetylaminodesthio-CoA. Consistent with these results was the demonstration of an acetyl-enzyme compound when the thiolase was incubated with [1-¹⁴C]acetyl-CoA. The sensitivity of the acetyl-enzyme bond to borohydride reduction and the protection afforded by acyl-CoA substrates against enzyme inactivation by sulfhydryl reagents indicated that acetyl groups are bound to the enzyme by a thiolester bond.     

Crystallization and preliminary crystallographic analysis at low resolution of the allosteric l-lactate dehydrogenase from Lactobacillus casei

The allosteric l-lactate dehydrogenase from Lactobacillus casei has been crystallized in its complex with the activators fructose-1,6-diphosphate and Co²⁺. The enzyme crystallizes in space group C2 with six tetramers in the unit cell. At very low resolution, 00l reflexions are absent for l ≠ 3n. The orientation of the molecular axes has been determined using the rotation function. All tetramers in the unit cell exhibit excellent 222 symmetry, and the overall arrangement resembles the packing that would be expected in the higher symmetry space group P3₁21. Comparison with the apo-enzyme structure of M4-lactate dehydrogenase from dogfish indicates high structural similarity between these enzymes and allowed us to identify the molecular axes of L. caseil-lactate dehydrogenase in terms of the “standard” molecular co-ordinate system P, Q, R. The similarity of both enzymes is good enough to allow the structure determination of L. caseil-lactate dehydrogenase by molecular replacement using the dogfish enzyme as a model.Sequencing results show that L. caseil-lactate dehydrogenase is lacking the N-terminal arm of vertebrate lactate dehydrogenases and electron density maps at 5 Å resolution indicate that ligands might possibly bind in the region of the missing arm. The active site loop is involved in intermolecular contacts and its structure might be different from both, apo- and ternary dogfish l-lactate dehydrogenase.