Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases

Prabhakar L Srivastava; Sam T Johns; Rebecca Walters; David J Miller; Marc W Van der Kamp; Rudolf K Allemann

doi:10.1021/acscatal.3c03920

Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases

ACS Catal. 2023 Oct 20;13(21):14199-14204. doi: 10.1021/acscatal.3c03920. eCollection 2023 Nov 3.

Authors

Prabhakar L Srivastava¹, Sam T Johns², Rebecca Walters², David J Miller¹, Marc W Van der Kamp², Rudolf K Allemann¹

Affiliations

¹ School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, United Kingdom.
² School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, United Kingdom.

Abstract

Terpene synthases (TS) catalyze complex reactions to produce a diverse array of terpene skeletons from linear isoprenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchoulol. Using simulation-guided engineering, we obtained PTS variants that eliminate water capture. Further, we demonstrate that modifying the structurally conserved Hα-1 loop also reduces hydroxylation in PTS, as well as in germacradiene-11-ol synthase (Gd11olS), leading to cyclic neutral intermediates as products, including α-bulnesene (PTS) and isolepidozene (Gd11olS). Hα-1 loop modification could be a general strategy for engineering sesquiterpene synthases to produce complex cyclic hydrocarbons without the need for structure determination or modeling.