Bioactive C-geranylated metabolites from Piper crassinervium: biological and biosynthetic studies

Abstract

Piperaceae family comprises 4 genera and more than 4000 species being the Piper genus the most abundant with approximately 2000 species [1]. Piper crassinervium accumulates antifungal, trypanocidal and antioxidant geranylated metabolites derived from p-hydroquinone and benzoic acid [2,3]. Aromatic geranyltransferases are a group of geranyltransferases that catalyze the C-geranylation step on aromatic substrates; they are membrane-bound enzymes and have showed strict substrate specificity for geranyldiphosphate (GPP) as the prenyl donor, although accept a variety of substrates as acceptors of GPP moieties [4,5]. Here we report the in vivo and in vitro biosynthetic studies carried out to elucidate the biosynthetic origin of geranyl moiety in compounds 1 and 2. The pattern of incorporation of [1-13C]-D-glucose determined by quantitative 13C NMR spectroscopy indicated that geranyl moiety come from mevalonate and triose/piruvate pathways in compound 1, but only the triose/piruvate pathway appear involved in compound 2. In addition, in vitro enzymatic studies of proteic extracts from leaves of P. crassinervium with 4-hydroxy and 3,4-dihydroxy benzoic acids as substrates, showed that O-geranylated products were biosynthesized instead of the C-geranylated ones. Purification and characterization processes of geranyltransferase activity, involving 1D, 2D SDS PAGE and MS studies of microsomal fractions are also discussed.