A 2-oxoglutarate-dependent Dioxygenase From Ruta Graveolens L. Exhibits P-Coumaroyl CoA 2'-hydroxylase Activity (C2'H): A Missing Step in the Synthesis of Umbelliferone in Plants

Plant J. 2012 May;70(3):460-70. doi: 10.1111/j.1365-313X.2011.04879.x. Epub 2012 Jan 16.

Abstract

Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6'-hydroxylase activity (Bourgaud et al., 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6'-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2'-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Conserved Sequence
  • Coumarins / analysis
  • Coumarins / isolation & purification
  • Coumarins / metabolism
  • Dioxygenases / antagonists & inhibitors
  • Dioxygenases / genetics
  • Dioxygenases / isolation & purification
  • Dioxygenases / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Furocoumarins / metabolism
  • Furocoumarins / pharmacology
  • Gene Expression / genetics
  • Molecular Sequence Data
  • Phylogeny
  • Plant Leaves / chemistry
  • Plant Leaves / enzymology
  • Plant Leaves / genetics
  • Plant Proteins / antagonists & inhibitors
  • Plant Proteins / genetics
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism
  • Plant Roots / chemistry
  • Plant Roots / enzymology
  • Plant Roots / genetics
  • Plant Stems / chemistry
  • Plant Stems / enzymology
  • Plant Stems / genetics
  • RNA, Plant / metabolism
  • Ruta / chemistry
  • Ruta / enzymology*
  • Ruta / genetics
  • Scopoletin / analysis
  • Scopoletin / metabolism
  • Sequence Alignment
  • Tobacco / enzymology
  • Tobacco / genetics
  • Transgenes
  • Umbelliferones / analysis
  • Umbelliferones / biosynthesis*
  • Umbelliferones / metabolism

Substances

  • Coumarins
  • Furocoumarins
  • Plant Proteins
  • RNA, Plant
  • Umbelliferones
  • 7-hydroxycoumarin
  • Dioxygenases
  • Scopoletin