Browsing by Subject "Betalamic acid"

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    Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains
    (2012-01-24) Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular A
    Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors to flowers, fruits and other parts of most plants of the order Caryophyllales. The formation of the structural unit of all betalains, betalamic acid from the precursor amino acid 4,5-dihydroxyphenylalanine is catalyzed by the enzyme 4,5-DOPAextradiol-dioxygenase followed by intramolecular cyclization of the 4,5-secodopa intermediate. This paper describes the purification and the molecular and functional characterization of an active 4,5-DOPA-extradiol-dioxygenase from the best-known source of betalains—Beta vulgaris—after heterologous expression in Escherichia coli. The enzyme is a monomeric protein with a molecular mass of 32 kDa characterized by chromatography, electrophoresis and MALDITOF analysis. Enzyme kinetic properties are characterized in the production of betalamic acid, the structural, chromophoric and bioactive unit of plant pigment betalains.
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    Escherichia coli protein YgiD produces the structural unit of plant pigments betalains: characterization of a prokaryotic enzyme with DOPA-extradiol-dioxygenase activity
    (Springer, 2013-05-12) Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular "A"
    Betalamic acid is the structural unit of all the natural pigments betalains. These are nitrogen-containing water soluble compounds with high colorant and bioactive properties, characteristic of plants of the order Caryophyllales. The formation of betalamic acid from the precursor amino acid 3,4-dihydroxy-L-phenylalanine (L-DOPA) by the enzyme 4,5- DOPA-extradiol-dioxygenase was supposed to be restricted to plants of this order and two fungal species. Here, the first case of betalamic acid formation by an enzyme other than eukaryotes is reported with a homolog enzyme from Escherichia coli. The protein YgiD has been cloned, expressed, and purified to carry out its molecular and functional characterization. The enzyme was obtained as a monomeric active protein with a molecular mass of 32 kDa characterized by chromatography, electrophoresis, and MALDI-TOF analysis. Enzyme kinetic properties are characterized in the transformation of the relevant substrate L-DOPA. Reaction was analyzed spectrophotometrically and by HPLC-DAD, electrospray ionization mass spectrometry, and time-of-flight mass spectrometry
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    First Betalain-producing bacteria break the exclusive presence of the pigments in the plant kingdom
    (American Society for Microbiology, 2019-03-19) Contreras Llano, Luis Eduardo; Guerrero Rubio, María Alejandra; Lozada Ramírez, José Daniel; Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular A
    The biosynthesis of antioxidant pigments, namely, betalains, was believed to be restricted to Caryophyllales plants. This paper changes this paradigm, and enzyme mining from bacterial hosts promoted the discovery of bacterial cultures producing betalains. The spectrum of possible sources of betalain pigments in nature is broadened by our description of the first betalain-forming bacterium, Gluconacetobacter diazotrophicus. The enzyme-specific step is the extradiol cleavage of the precursor amino acid L-dihydroxyphenylalanine (L-DOPA) to form the structural unit betalamic acid. Molecular and functional work conducted led to the characterization of a novel dioxygenase, a polypeptide of 17.8 kDa with a Km of 1.36 mM, with higher activity and affinity than those of its plant counterparts. Its superior activity allowed the first experimental characterization of the early steps in the biosynthesis of betalains by fully characterizing the presence and time evolution of 2,3- and 4,5- seco-DOPA intermediates. Furthermore, spontaneous chemical reactions are characterized and incorporated into a comprehensive enzymatic-chemical mechanism that yields the final pigments. Several studies have demonstrated the health-promoting effects of betalains due to their high antioxidant capacity and their positive effect on the dose-dependent inhibition of cancer cells and their proliferation. To date, betalains were restricted to plants of the order Caryophyllales and some species of fungi, but the present study reveals the first betalain-producing bacterium, as well as the first steps in the formation of pigments. This finding demonstrates that betalain biosynthesis can be expanded to prokaryotes.
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    Fluorescent Bioinspired Protein Labeling with Betalamic Acid. Derivatization and Characterization of Novel Protein-Betaxanthins
    (2016-10) Francisco, García-Carmona; Mercedes, Jiménez-Atiénzar; Cabanes Cos, Juana; Escribano Cebrián, Josefa; Gandía Herrero, Fernando; Bioquímica y Biología Molecular A
    Betaxanthins are the water-soluble pigments that bestow yellow coloration to fruits, flowers and roots of plants of the Caryophyllales order and present autofluorescence after excitation with blue light. In this work, the semi-synthesis of betaxanthins derived from macromolecules is achieved for the first time by exploiting the reactivity of amine groups belonging to proteins. The synthesis of protein-betaxanthins is demonstrated by spectrophotometry and HPLC-ESI-TOF-MS mass analysis. The derivatization with betalamic acid was in a ratio 1:1 and yielded protein-betaxanthins yellow in color that exhibited fluorescent properties with a maximum excitation wavelength of 476 nm and a maximum emission wavelength of 551 nm. Moreover, staining can be started from purified betalamic acid or directly from raw red beet root extracts. The novel bioinspired labeling reaction allowed protein detection in conventional fluorescence scanning and imaging systems and opens a new perspective for betalamic acid derived molecules as fluorescent probes with multiple biological applications.
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    One-Step synthesis of betalains using a novel betalamic acid derivatized support
    (American Chemical Society, 2014-04-01) Jiménez-Atiénzar, Mercedes; Cabanes Cos, Juana; Escribano Cebrián, Josefa; Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular "A"
    Betalains are plant pigments with high antioxidant and cancer hemopreventive properties used by the food industry as safe colorants. Betalains are restricted to species of the order Caryophyllales, and difficulty in obtaining individual molecules has limited their structural identification and application. This study was designed to develop a betalamic acid derivatized support generated from a primary amine polymer. The novel material presents color properties of a pseudobetaxanthin, and it is stable for at least 6 months. The bond formed can be displaced at mild conditions by the addition of amines in aqueous solutions over a broad pH range and at 25 °C. This releases the betalamic acid while forming the corresponding pigment. This one-step procedure significantly simplifies the process of obtaining semisynthetic betalains, and it is optimized here for the formation of betaxanthins and betacyanins derived from tyramine, dopamine, pyrrolidine, and indoline. The new method makes access to single betalains available to the entire scientific community and could stimulate research and applications in the field.