Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis

Biochem Biophys Res Commun. 2013 Feb 15;431(3):597-603. doi: 10.1016/j.bbrc.2012.12.129. Epub 2013 Jan 9.

Abstract

Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzophenanthridines / biosynthesis*
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / classification
  • Cytochrome P-450 Enzyme System / genetics*
  • DNA, Complementary / genetics*
  • DNA, Complementary / isolation & purification
  • Isoquinolines
  • Opium*
  • Papaver / enzymology*
  • Papaver / genetics
  • Phylogeny
  • Plant Proteins / chemistry
  • Plant Proteins / classification
  • Plant Proteins / genetics*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / classification
  • Recombinant Proteins / genetics
  • Saccharomyces cerevisiae
  • Substrate Specificity

Substances

  • Benzophenanthridines
  • DNA, Complementary
  • Isoquinolines
  • Plant Proteins
  • Recombinant Proteins
  • Opium
  • Cytochrome P-450 Enzyme System
  • sanguinarine
  • (S)-cis-N-methylstylopine 14-hydroxylase, Papaver somniferum