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Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains

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Abstract

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-DOPA-extradiol-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 MALDI-TOF 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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Abbreviations

AA:

Ascorbic acid

DODA:

4,5-DOPA-extradiol-dioxygenase

DOPA:

Dihydroxyphenylalanine

EDTA:

Ethylenediaminetetraacetic acid

ESI-MS:

Elecrospray ionization mass analysis

IPTG:

Isopropyl-1-thio-β-d-galactopyranoside

LB:

Luria broth

MALDI-TOF:

Matrix-assisted laser desorption/ionization-time of flight

PDA:

Photodiode array

TFA:

Trifluoroacetic acid

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Acknowledgments

This work was supported by the Ministerio de Ciencia e Innovación (MCINN, FEDER, Spain) (Project AGL2010-17938), and by Programa de Ayudas a Grupos de Excelencia de la Región de Murcia, of the Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia (Plan Regional de Ciencia y Tecnología 2007/2010). F. Gandía-Herrero holds a contract with the “Programa Ramón y Cajal” (MICINN, FEDER, Spain). The authors are grateful to Sofía Jiménez for help in gel filtration analysis and to Alejandro Torrecillas (SAI, University of Murcia) for skillful technical assistance in MALDI-TOF experiments.

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  1. Departamento de Bioquímica y Biología Molecular A, Unidad Docente de Biología, Facultad de Veterinaria, Universidad de Murcia, 30100, Espinardo, Murcia, Spain

    Fernando Gandía-Herrero & Francisco García-Carmona

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  1. Fernando Gandía-Herrero
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  2. Francisco García-Carmona
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Correspondence to Fernando Gandía-Herrero.

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Supplementary Fig. 1 Beta vulgaris 4,5-DODA gene sequence, as deposited in NCBI and modified for this work. The amino acid equivalence is shown.

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Gandía-Herrero, F., García-Carmona, F. Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains. Planta 236, 91–100 (2012). https://doi.org/10.1007/s00425-012-1593-2

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