Structure-Guided Engineering of a Bacterial Sesterterpene Synthase for Sesterviridene Diversification

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Terpene synthases are crucial enzymes generating diverse molecular structures from simple precursors via carbocation cascades.
• Understanding the structural basis of terpene synthase activity is key to controlling product formation.

Purpose of the Study:

• To determine the crystal structure of the bacterial sesterterpene synthase StvirS.
• To investigate the role of the active site in controlling terpene biosynthesis through structure-based engineering.
• To discover novel sesterterpenes by modifying StvirS.

Main Methods:

• X-ray crystallography was used to obtain the structure of StvirS bound to geranylfarnesyl thiopyrophosphate (GFSPP).
• Site-directed mutagenesis was employed to engineer 23 variants of StvirS at 11 positions.
• Product analysis involved isotopic labeling to confirm reaction pathways and determine absolute configurations.

Main Results:

• The crystal structure revealed a preorganized active site in StvirS, guiding the folding of the C25 backbone.
• Engineering StvirS resulted in 23 variants, producing 13 previously unknown sesterterpenes.
• Mutations altered reaction pathways, stereochemistry, and product termination, highlighting enzyme sensitivity.

Conclusions:

• Precise noncovalent interactions within the StvirS active site govern terpene biosynthesis.
• Structure-based enzyme engineering is a powerful strategy for reprogramming terpene cyclase reactivity and discovering new compounds.