L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production

l-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous l-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of l-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess l-DOPA, both the betaxanthin and hygroaurin adducts of l-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other l-DOPA dioxygenases. The rate constants for the conversion of l-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.

[1]  F. Forti,et al.  A metal-free blue chromophore derived from plant pigments , 2020, Science Advances.

[2]  E. L. Bastos,et al.  Development of a Photoinduced Fragmentation Ion Trap for Infrared Multiple Photon Dissociation Spectroscopy. , 2020, Rapid communications in mass spectrometry : RCM.

[3]  Tao Feng,et al.  The evolution of betalain biosynthesis in Caryophyllales. , 2019, The New phytologist.

[4]  Z. Zhang,et al.  Functional characterization of the horizontally transferred 4,5‐DOPA extradiol dioxygenase gene in the domestic silkworm, Bombyx mori , 2019, Insect molecular biology.

[5]  F. García-Carmona,et al.  First Betalain-Producing Bacteria Break the Exclusive Presence of the Pigments in the Plant Kingdom , 2019, mBio.

[6]  K. Schwinn,et al.  Functions of Flavonoid and Betalain Pigments in Abiotic Stress Tolerance in Plants , 2018 .

[7]  Huimin Zhao,et al.  Recent developments in the application of P450 based biocatalysts. , 2018, Current opinion in chemical biology.

[8]  Michael T. McManus,et al.  Annual Plant Reviews , 2018 .

[9]  A. Aharoni,et al.  "La Vie en Rose": Biosynthesis, Sources, and Applications of Betalain Pigments. , 2018, Molecular plant.

[10]  A. Osbourn.  Painting with betalains , 2017, Nature Plants.

[11]  Jiasong Li,et al.  Oxygen activation by mononuclear nonheme iron dioxygenases involved in the degradation of aromatics , 2017, JBIC Journal of Biological Inorganic Chemistry.

[12]  Alicia Gil-Ramírez,et al.  Mushrooms do not contain flavonoids , 2016 .

[13]  Long-Fei Wu,et al.  Ferrous iron and α-ketoglutarate-dependent dioxygenases in the biosynthesis of microbial natural products. , 2016, Biochimica et biophysica acta.

[14]  A. Aharoni,et al.  Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants. , 2016, The New phytologist.

[15]  J. Novotná,et al.  New Concept of the Biosynthesis of 4-Alkyl-L-Proline Precursors of Lincomycin, Hormaomycin, and Pyrrolobenzodiazepines: Could a γ-Glutamyltransferase Cleave the C–C Bond? , 2016, Front. Microbiol..

[16]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[17]  S. Rokita,et al.  Identification of the dioxygenase-generated intermediate formed during biosynthesis of the dihydropyrrole moiety common to anthramycin and sibiromycin. , 2015, Bioorganic & medicinal chemistry.

[18]  K. Davies.  Swapping one red pigment for another , 2014, Nature Genetics.

[19]  M. Vissers,et al.  Ascorbate as a Co-Factor for Fe- and 2-Oxoglutarate Dependent Dioxygenases: Physiological Activity in Tumor Growth and Progression , 2014, Front. Oncol..

[20]  F. García-Carmona,et al.  Escherichia coli protein YgiD produces the structural unit of plant pigments betalains: characterization of a prokaryotic enzyme with DOPA-extradiol-dioxygenase activity , 2014, Applied Microbiology and Biotechnology.

[21]  A. Bar‐Even,et al.  Engineering specialized metabolic pathways--is there a room for enzyme improvements? , 2013, Current opinion in biotechnology.

[22]  F. Wartchow,et al.  Taxonomic studies of Amanita muscaria (L.) Lam (Amanitaceae, Agaricomycetes) and its infraspecific taxa in Brazil , 2013 .

[23]  J. McGrath,et al.  The beet R locus encodes a new cytochrome P450 required for red betalain production , 2012, Nature Genetics.

[24]  Sung Gyun Kang,et al.  One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies , 2012, Applied and Environmental Microbiology.

[25]  F. García-Carmona,et al.  Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains , 2012, Planta.

[26]  Pamela S Soltis,et al.  Complex pigment evolution in the Caryophyllales. , 2011, The New phytologist.

[27]  T. Bugg,et al.  Non-heme iron-dependent dioxygenases: unravelling catalytic mechanisms for complex enzymatic oxidations. , 2008, Current opinion in chemical biology.

[28]  Á. Gil-Izquierdo,et al.  Betalains in the era of global agri-food science, technology and nutritional health , 2008, Phytochemistry Reviews.

[29]  Florian C. Stintzing,et al.  Betalains – emerging prospects for food scientists , 2007 .

[30]  Mudita Singhal,et al.  COPASI - a COmplex PAthway SImulator , 2006, Bioinform..

[31]  Luke E. Ulrich,et al.  Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility , 2006, Proceedings of the National Academy of Sciences.

[32]  Florian C. Stintzing,et al.  Betalain stability and degradation--structural and chromatic aspects , 2006 .

[33]  J. Bolin,et al.  The Ins and Outs of Ring-Cleaving Dioxygenases , 2006, Critical reviews in biochemistry and molecular biology.

[34]  F. García-Carmona,et al.  Botany: Floral fluorescence effect , 2005, Nature.

[35]  U. Hinz,et al.  Characterization and Functional Identification of a Novel Plant 4,5-Extradiol Dioxygenase Involved in Betalain Pigment Biosynthesis in Portulaca grandiflora , 2004, Plant Physiology.

[36]  T. Bugg.  Dioxygenase Enzymes: Catalytic Mechanisms and Chemical Models , 2003 .

[37]  D. Strack,et al.  Recent advances in betalain research. , 2003, Phytochemistry.

[38]  V. Wray,et al.  Formation and occurrence of dopamine-derived betacyanins. , 2001, Phytochemistry.

[39]  Kobayashi,et al.  The decisive step in betaxanthin biosynthesis is a spontaneous reaction1 , 1999, Plant physiology.

[40]  L. Mueller,et al.  Biochemical complementation of the betalain biosynthetic pathway in Portulaca grandiflora by a fungal 3,4-dihydroxyphenylalanine dioxygenase , 1997, Planta.

[41]  U. Hinz,et al.  The gene coding for the DOPA dioxygenase involved in betalain biosynthesis in Amanita muscaria and its regulation , 1997, Molecular and General Genetics MGG.

[42]  Lukas A. Mueller,et al.  The formation of betalamic acid and muscaflavin by recombinant dopa-dioxygenase from amanita. , 1997 .

[43]  H. Wyler,et al.  2,3- and 4,5-Secodopa, the Biosynthetic Intermediates Generated from L-Dopa by an Enzyme System Extracted from the Fly Agaric, Amanita muscaria L., and Their Spontaneous Conversion to Muscaflavin and Betalamic Acid, Respectively, and Betalains , 1991 .

[44]  E. Land,et al.  A pulse radiolysis investigation of the oxidation of the melanin precursors 3,4-dihydroxyphenylalanine (dopa) and the cysteinyldopas. , 1985, Biochimica et biophysica acta.

[45]  S. Albracht,et al.  Prolyl 4-hydroxylase activity in relation to the oxidation state of enzyme-bound iron. The role of ascorbate in peptidyl proline hydroxylation. , 1982, Biochimica et biophysica acta.

[46]  A. Kemp,et al.  The function of ascorbate with respect to prolyl 4-hydroxylase activity. , 1981, Biochimica et biophysica acta.

[47]  H. Musso,et al.  Über die Synthese des Muscaflavins. Vorläufige Mitteilung , 1979 .

[48]  K. Saito,et al.  Biosynthesis of stizolobinic acid and stizolobic acid in higher plants. , 1976, European journal of biochemistry.

[49]  M. Piattelli,et al.  A new betaxanthin from Glottiphyllum longum , 1973 .

[50]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[51]  Zachary N. Russ,et al.  Bioproduction of a betalain color palette in Saccharomyces cerevisiae. , 2018, Metabolic engineering.

[52]  John E. Haight,et al.  Molecular phylogeny, morphology, pigment chemistry and ecology in Hygrophoraceae (Agaricales) , 2013, Fungal Diversity.

[53]  T. Harris,et al.  Measurement of enzyme activity. , 2009, Methods in enzymology.

[54]  T. Bugg,et al.  Enzymatic cleavage of aromatic rings: mechanistic aspects of the catechol dioxygenases and later enzymes of bacterial oxidative cleavage pathways , 1998 .

[55]  H. A. Stafford.  Anthocyanins and betalains: evolution of the mutually exclusive pathways , 1994 .

[56]  P. Girod,et al.  BIOGENESIS OF BETALAINS : PURIFICATION AND PARTIAL CHARACTERIZATION OF DOPA 4,5-DIOXYGENASE FROM AMANITA MUSCARIA , 1991 .

[57]  G. F. Trezzini,et al.  Characterization of some natural and semi-synthetic betaxanthins , 1991 .

[58]  H. Musso.  The pigments of fly agaric, Amanita muscaria , 1979 .

[59]  K. Saito,et al.  Biosynthesis of stizolobinic acid and stizolobic acid in higher plants. An enzyme system(s) catalyzing the conversion of dihydroxyphenylalanine into stizolobinic acid and stizolobic acid from etiolated seedlings of Stizolobium hassjoo. , 1976, European journal of biochemistry.