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.

中文翻译：

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飞木耳菌的L-DOPA双加氧酶：dodA基因序列的修订和酶促色素生成的机理

L-DOPA外二醇双加氧酶（DODA）催化甜菜碱和潮红素颜料的产生。尽管甜菜碱是植物和真菌共同产生的，但在石竹叶科和担子菌科中发现的DODA序列并不保守。在这里，我们修改了飞木耳[Amanita muscaria（L.）Lam。]的dodA基因的编码区，并描述了下游的替代起始密码子，该密码子使得AmDODA（一种混杂的L-DOPA双加氧酶）能够异源表达。AmDODA比先前报道的重组DODA短43个氨基酸残基，但可催化两种异构的seco-DOPA的形成，它们是甜菜碱和潮红的生物合成前体。AmDODA的推定活性位点包含两个不同的His-His-Glu基序，这些基序可以根据针对非血红素铁依赖性双加氧酶提出的机理来解释L-DOPA的双重切割。加入过量的L-DOPA后，既产生了L-DOPA的虾青素又产生了潮红素加合物。pH 8.5时的酶催化动力学参数与其他L-DOPA双加氧酶的动力学参数相似。通过动力学模型估算了L-DOPA转化为Betalamic酸和Muscaflavin的速率常数，从而提出了形成颜料的机理。这些结果有助于理解细菌，真菌和植物色素的生物合成，为潮红素的生物技术生产，以及为环境修复开发更混杂的双加氧酶。