Molecular Cloning and Characterization of a Cytochrome P450 in Sanguinarine Biosynthesis From Eschscholzia Californica Cells

Phytochemistry. 2013 Jul;91:100-8. doi: 10.1016/j.phytochem.2012.02.013. Epub 2012 Mar 13.

Abstract

Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Benzophenanthridines / biosynthesis*
  • Benzophenanthridines / chemistry
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / genetics*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Eschscholzia / chemistry
  • Eschscholzia / cytology
  • Eschscholzia / metabolism*
  • Isoquinolines / chemistry
  • Molecular Sequence Data
  • Molecular Structure
  • Sequence Alignment
  • Substrate Specificity

Substances

  • Benzophenanthridines
  • Isoquinolines
  • Cytochrome P-450 Enzyme System
  • sanguinarine

Associated data

  • GENBANK/AB598834