From functional plasticity of two diterpene synthases (IrTPS2/IrKSL3a) to enzyme evolution

Area of Science:

• Biochemistry
• Natural Product Chemistry
• Enzymology

Background:

• Terpenoids are diverse natural products synthesized by terpene synthases (TPSs).
• TPS enzymes catalyze complex carbocationic cascade reactions, but the quenching mechanisms are often unclear.
• IrKSL3a and IrTPS2 from Isodon rubescens, despite high sequence homology, exhibit different catalytic strategies.

Purpose of the Study:

• To elucidate the mechanism of water addition in IrTPS2 for nezukol formation.
• To investigate the role of specific amino acid residues in TPS catalytic activity and product specificity.
• To engineer IrKSL3a for novel product generation based on mechanistic insights.

Main Methods:

• Site-directed mutagenesis of key residues in IrTPS2.
• Multiscale quantum mechanics/molecular mechanics (QM/MM) simulations.
• Enzymatic assays with engineered variants and phylogenetically diverse TPSs.

Main Results:

• Identified a threonine and serine residue in IrTPS2 crucial for hydrogen-bonding and water addition.
• Demonstrated that steric hindrance from a β-methyl side chain can block water binding.
• Successfully engineered IrKSL3a to produce nezukol and other hydroxylated products.
• Showed that identified residues are necessary but not sufficient for efficient water addition in other kaurene synthase-like (KSL) TPSs.

Conclusions:

• Specific amino acid residues dictate the distinct catalytic mechanisms of closely related TPS enzymes.
• Mechanistic understanding allows for enzyme engineering to alter product profiles.
• Evolutionary pressures have shaped TPS enzymes for specific product formation, such as nezukol synthesis.