Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases.

Area of Science:

• Biochemistry
• Enzyme Engineering
• Natural Product Synthesis

Background:

• Terpene synthases (TS) are crucial enzymes that synthesize diverse terpene structures from linear precursors.
• Patchoulol synthase (PTS) from Pogostemon cablin specifically produces patchoulol from farnesyl diphosphate.
• Controlling the hydroxylation activity of TS is key to generating specific terpene skeletons.

Purpose of the Study:

• To engineer terpene synthases, specifically PTS, to eliminate water capture and reduce hydroxylation.
• To investigate the role of the Hα-1 loop in controlling TS activity and product formation.
• To develop a generalizable strategy for engineering sesquiterpene synthases to produce cyclic hydrocarbons.

Main Methods:

• Simulation-guided engineering was employed to modify PTS.
• Site-directed mutagenesis was used to alter the conserved Hα-1 loop in PTS and germacradiene-11-ol synthase (Gd11olS).
• Product analysis was performed to identify the resulting terpene skeletons.

Main Results:

• Engineered PTS variants were generated that successfully eliminated water capture.
• Modification of the Hα-1 loop in both PTS and Gd11olS significantly reduced hydroxylation.
• This modification led to the production of cyclic neutral hydrocarbon intermediates, including α-bulnesene from PTS and isolepidozene from Gd11olS.

Conclusions:

• Hα-1 loop modification is an effective strategy for engineering sesquiterpene synthases to control hydroxylation.
• This approach allows for the production of complex cyclic hydrocarbons without requiring prior structure determination or modeling.
• The findings offer a generalizable method for tailoring TS activity for novel terpene synthesis.