Structural insights into γ-presodorifen pyrophosphate synthase

Area of Science:

• Biochemistry
• Structural Biology
• Metabolic Engineering

Background:

• Canonical terpenoids follow the (C5H8)n formula, but non-canonical variants breaking the isoprene rule are emerging.
• C17 homosesquiterpenoid chlororaphens originate from C15 farnesyl pyrophosphate (FPP) via methyltransferases (MTs).
• A bifunctional S-adenosyl-L-methionine (SAM)-dependent MT initiates this by methylating FPP at C10, triggering cyclization to C16 γ-presodorifen pyrophosphate (γ-PSPP).

Purpose of the Study:

• To determine the crystal structures of γ-PSPP synthases from Variovorax boronicumulans PHE5-4 (VbFPPMT) and Pseudomonas chlororaphis O6 (PcFPPMT).
• To investigate the structural basis for the catalytic mechanism of these bifunctional methyltransferases.
• To provide a framework for engineering C16/C17 terpenoid biosynthesis.

Main Methods:

• X-ray crystallography to obtain structures of γ-PSPP synthases and substrate-bound complexes.
• Molecular docking to predict substrate-enzyme interactions.
• Site-directed mutagenesis to identify key catalytic residues.

Main Results:

• Crystal structures of VbFPPMT and PcFPPMT, including a Mg2+- and geranyl pyrophosphate (GPP)-bound complex of PcFPPMT, were determined.
• The structures revealed the overall enzyme architecture, catalytic center composition, and a flexible region for substrate and cofactor binding.
• Key residues governing the catalytic mechanism of these bifunctional γ-PSPP synthases were identified through integrated structural and biochemical analyses.

Conclusions:

• The study elucidates the structural mechanisms of non-canonical methyltransferases involved in terpenoid biosynthesis.
• These findings offer crucial insights into the catalytic strategies of enzymes producing novel terpenoid structures.
• The established framework supports rational engineering of pathways for producing C16/C17 terpenoids.