Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut.

Stilbene synthase ( resveratrol -forming) converts one molecule of rho- coumaroyl -CoA and three molecules of malonyl-CoA into 3,4',5- trihydroxystilbene . Following selective induction of stilbene synthesis in cell suspension cultures of peanut (Arachis hypogaea), the enzyme was extracted and purified to apparent homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. The enzyme was found to be a dimer of estimated Mr = 90,000 exhibiting under denaturing conditions a subunit Mr of approximately 45,000. The isoelectric point was determined with pI = 4.8. The enzyme's high selectivity towards rho- coumaroyl -CoA (Km = 2 microM) as substrate qualified it as resveratrol -forming stilbene synthase. Structurally related CoA esters, e.g. dihydro-rho- coumaroyl -CoA and cinnamoyl-CoA, were converted less than 1/10 as efficiently as rho- coumaroyl -CoA. Malonyl-CoA (Km = 10 microM) could not be substituted by acetyl-CoA. The purified enzyme was free of chalcone synthase activity. Antibodies raised against stilbene synthase were shown to be monospecific and not to cross-react with chalcone synthase.

9,10-Dihydrophenanthrenes as phytoalexins of Orchidaceae. Biosynthetic studies in vitro and in vivo proving the route from L-phenylalanine to dihydro-m-coumaric acid, dihydrostilbene and dihydrophenanthrenes.

Hydroxy derivatives of 9,10-dihydrophenanthrenes, orchinol and hircinol, were isolated from bulbs of Orchidaceae which had been induced to accumulate phytoalexins. Incorporation of radioactive precursors, L-phenylalanine and various hydroxycinnamic acids, has been investigated by feeding experiments in vivo. m-Coumaric acid and dihydro-m-coumaric acid were found to be efficiently incorporated into the dihydrophenanthrene derivatives. Dihydro-m-coumaric acid was not only converted into the dihydrophenanthrenes but was also formed from L-phenylalanine in the same tissue; it was thus proved to be an intermediate. The role of dihydro-m-coumaric acid was substantiated by studies in vitro. An active stilbene synthase was detected in enzyme preparations from induced orchid bulbs and assayed with different CoA esters. The enzyme, characterized on the basis of its substrate specificity, selectively converted dihydro-m-coumaroyl-CoA plus malonyl-CoA into 3,3',5-trihydroxybibenzyl. The role of 3,3',5-trihydroxybibenzyl as physiological intermediate was further corroborated by investigations with intact plants. Both its formation from phenylpropanoids and its conversion into orchinol was demonstrated. The data provided evidence for a biosynthetic sequence originating from L-phenylalanine and leading to 9,10-dihydrophenanthrenes via m-coumaric acid, dihydro-m-coumaric acid, and 3,3',5-trihydroxybibenzyl.