Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants.
Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family--as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.
Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom.
Katrin
K
Geisler
Richard K
RK
Hughes
Frank
F
Sainsbury
George P
GP
Lomonossoff
Martin
M
Rejzek
Shirley
S
Fairhurst
Carl-Erik
CE
Olsen
Mohammed Saddik
MS
Motawia
Rachel E
RE
Melton
Andrew M
AM
Hemmings
Søren
S
Bak
Anne
A
Osbourn
D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000378:metabolism*; D018554:Avena / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D058886:Sterol 14-Demethylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology; D014315:Triterpenes / Q000378:metabolism*
D000890:Anti-Infective Agents; D014315:Triterpenes; D058886:Sterol 14-Demethylase; D019751:Intramolecular Transferases; C039924:2,3-oxidosqualene-beta-amyrin-cyclase
CPMV-HT transient expression; cytochrome P450 monooxygenase CYP51 family; disease resistance; neofunctionalization; terpenes