A novel 4-hydroxycoumarin biosynthetic pathway.

Area of Science:

• Biochemistry
• Plant Science
• Enzymology

Background:

• Coumarin formation occurs in plants with melilotoside upon cell damage.
• Fungal conversion of trans-ortho-coumaric acid yields 4-hydroxycoumarin, leading to dicoumarol, an anticoagulant precursor.
• Dicoumarol is the forerunner of warfarin anticoagulants.

Purpose of the Study:

• To investigate the role of biphenyl synthase (BIS) in 4-hydroxycoumarin formation.
• To characterize novel BIS isoenzymes from Sorbus aucuparia.
• To explore the enzymatic pathway for 4-hydroxycoumarin synthesis.

Main Methods:

• Isolation of two new biphenyl synthase (BIS) cDNAs from elicitor-treated Sorbus aucuparia cell cultures.
• Enzymatic assays using ortho-hydroxybenzoyl-CoA and malonyl-CoA to assess 4-hydroxycoumarin production.
• Feeding experiments with N-acetylcysteamine thioester of salicylic acid in plant cell cultures.

Main Results:

• Two BIS isoenzymes were identified, preferring ortho-hydroxybenzoyl-CoA as a starter substrate.
• These isoenzymes catalyzed the formation of 4-hydroxycoumarin via decarboxylative condensation with malonyl-CoA.
• 4-hydroxycoumarin accumulated in culture medium when elicitor-treated cells were supplied with salicylic acid derivatives.
• Incubation with benzoyl-CoA and malonyl-CoA yielded 3,5-dihydroxybiphenyl, a phytoalexin precursor.

Conclusions:

• Biphenyl synthase (BIS) is directly involved in the formation of 4-hydroxycoumarin.
• This enzymatic pathway provides a novel route for 4-hydroxycoumarin synthesis in plants.
• The findings contribute to understanding plant secondary metabolism and the biosynthesis of anticoagulant precursors.