Stilbene synthase from Scots pine (Pinus sylvestris)

Area of Science:

• Plant Biochemistry
• Enzyme Kinetics
• Molecular Biology

Background:

• Stilbene synthases (StS) are classified by substrate preference: cinnamoyl-CoA preferring enzymes are pinosylvin synthases (PINS), and phenylpropionyl-CoA preferring enzymes are dihydropinosylvin synthases (DHPS).
• A previously cloned Scots pine (Pinus sylvestris) stilbene synthase was proposed to be a DHPS, with evidence for a second PINS.

Purpose of the Study:

• To re-evaluate the substrate preference and identity of the cloned Scots pine stilbene synthase.
• To investigate factors influencing enzyme activity and substrate specificity in heterologous expression systems.

Main Methods:

• Expression of the cloned stilbene synthase in E. coli.
• Kinetic analysis of enzyme activity with different substrates (cinnamoyl-CoA and phenylpropionyl-CoA).
• Mixing experiments using plant extracts from Pinus sylvestris and the expressed enzyme.

Main Results:

• Bacterial factors in E. coli initially affected substrate preference, but improved expression systems revealed cinnamoyl-CoA as the preferred substrate.
• Plant extracts from Pinus sylvestris contained factors that selectively altered substrate preference, explaining previous discrepancies.
• The cloned enzyme demonstrated characteristics of a pinosylvin synthase, with no evidence for a second stilbene synthase in the studied extracts.

Conclusions:

• The cloned enzyme from Scots pine is confirmed as a pinosylvin synthase.
• Heterologous expression systems and plant extracts can contain factors that interfere with the accurate functional characterization of plant enzymes.
• Careful consideration of host factors is crucial for the correct identification of cloned plant enzyme sequences.