Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis

Biochem J. 2013 Sep 1;454(2):209-16. doi: 10.1042/BJ20130574.

Abstract

Natural products biosynthesis often requires the action of multiple CYPs (cytochromes P450), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we characterized CYPs that catalyse alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, CYP76M6 and CYP76M8 were all shown to carry out C-7β hydroxylation, whereas CYP701A8 catalyses C-3α hydroxylation, with oxy groups found at both positions in oryzalexins A-D, suggesting that these may act consecutively in oryzalexin biosynthesis. In the present paper, we report that, although CYP701A8 only poorly reacts with 7β-hydroxy-ent-sandaracopimaradiene, CYP76M6 and CYP76M8 readily react with 3α-hydroxy-ent-sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C-9β by CYP76M6 or C-7β by CYP76M8, on different sides of the core tricyclic ring structure. Thus CYP76M6 and CYP76M8 have distinct non-redundant roles in orzyalexin biosynthesis. Moreover, the resulting 3α,7β- and 3α,9β-diols correspond to oryzalexins D and E respectively. Accordingly, the results of the present study complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalysed by CYP76M6 following C-3α hydroxylation has some implications for its active-site configuration, offering further molecular insight.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / metabolism*
  • Binding, Competitive
  • Catalytic Domain
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Diterpenes / chemistry
  • Diterpenes / metabolism*
  • Endoplasmic Reticulum / enzymology
  • Endoplasmic Reticulum / metabolism
  • Hydrogen Bonding
  • Hydroxylation
  • Isoenzymes / metabolism
  • Kinetics
  • Oryza / enzymology*
  • Oryza / metabolism
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Plastids / enzymology
  • Plastids / metabolism
  • Protein Conformation
  • Protein Transport
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sesquiterpenes / chemistry
  • Sesquiterpenes / metabolism
  • Stereoisomerism

Substances

  • Anti-Bacterial Agents
  • Diterpenes
  • Isoenzymes
  • Plant Proteins
  • Recombinant Proteins
  • Sesquiterpenes
  • ent-sandaracopimaradiene
  • oryzalexin D
  • oryzalexin E
  • phytoalexins
  • Cytochrome P-450 Enzyme System