Molecular Cloning and Characterization of CYP80G2, a Cytochrome P450 That Catalyzes an Intramolecular C-C Phenol Coupling of (S)-reticuline in Magnoflorine Biosynthesis, From Cultured Coptis Japonica Cells

J Biol Chem. 2008 Apr 4;283(14):8810-21. doi: 10.1074/jbc.M705082200. Epub 2008 Jan 29.

Abstract

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism
  • Amino Acid Sequence
  • Aporphines / chemistry
  • Aporphines / metabolism*
  • Benzylisoquinolines / chemistry
  • Benzylisoquinolines / metabolism*
  • Berberis / enzymology
  • Berberis / genetics
  • Cloning, Molecular
  • Coptis / cytology
  • Coptis / enzymology*
  • Coptis / genetics
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Molecular Sequence Data
  • NADP / genetics
  • NADP / metabolism
  • Oxygen / metabolism
  • Phenol / chemistry
  • Phenol / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid

Substances

  • Aporphines
  • Benzylisoquinolines
  • Recombinant Proteins
  • cytochrome P-450 CYP80A1 (Berberis stolonifera)
  • Phenol
  • NADP
  • Cytochrome P-450 Enzyme System
  • Alcohol Oxidoreductases
  • magnoflorine
  • Oxygen
  • reticuline

Associated data

  • GENBANK/AB288053