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

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

Cardiff University
University Walk

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Pages (from-to)	14199-14204
Number of pages	6
Journal	ACS Catalysis
Volume	13
Issue number	21
Early online date	20 Oct 2023
DOIs	https://doi.org/10.1021/acscatal.3c03920
Publication status	Published - 3 Nov 2023
Externally published	Yes

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10.1021/acscatal.3c03920Licence: CC BY

Cite this

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title = "Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases",

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.",

author = "Srivastava, \{Prabhakar L\} and Johns, \{Sam T\} and Rebecca Walters and Miller, \{David J\} and \{Van der Kamp\}, \{Marc W\} and Allemann, \{Rudolf K\}",

note = "{\textcopyright} 2023 The Authors. Published by American Chemical Society.",

year = "2023",

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doi = "10.1021/acscatal.3c03920",

language = "English",

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T1 - Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases

AU - Srivastava, Prabhakar L

AU - Johns, Sam T

AU - Walters, Rebecca

AU - Miller, David J

AU - Van der Kamp, Marc W

AU - Allemann, Rudolf K

PY - 2023/11/3

Y1 - 2023/11/3

N2 - 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.

AB - 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.

U2 - 10.1021/acscatal.3c03920

DO - 10.1021/acscatal.3c03920

M3 - Article

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SN - 2155-5435

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EP - 14204

JO - ACS Catalysis

JF - ACS Catalysis

IS - 21

ER -

Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases

Abstract

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