Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases

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

^*Corresponding author for this work

School of Biochemistry

Research output: Contribution to journal › Letter (Academic Journal) › peer-review

14 Citations (Scopus)

Abstract

Terpene synthases (TS) catalyse complex reactions to produce a diverse array of terpene skeletons from linear iso-prenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchou-lol. 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 sesquiter-pene 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

Bibliographical note

Funding Information:
This work was supported by the BBSRC (BB/R001596/1 and BB/R001332/1). M.W.V.d.K. was a BBSRC David Phillips Fellow (BB/M026280/1). Simulations were performed using the computational facilities of the Advanced Computing Research Centre, University of Bristol.

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

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Multi-scale enzyme modelling for SynBio: optimizing biocatalysts for selective synthesis of bioactive compounds
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@article{0a5f97f685ea40cfbdfff77d010d3dff,

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

abstract = "Terpene synthases (TS) catalyse complex reactions to produce a diverse array of terpene skeletons from linear iso-prenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchou-lol. 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 sesquiter-pene synthases to produce complex cyclic hydrocarbons, without the need for structure determination or modeling.",

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

note = "Funding Information: This work was supported by the BBSRC (BB/R001596/1 and BB/R001332/1). M.W.V.d.K. was a BBSRC David Phillips Fellow (BB/M026280/1). Simulations were performed using the computational facilities of the Advanced Computing Research Centre, University of Bristol. Publisher Copyright: {\textcopyright} 2023 The Authors. Published by American Chemical Society.",

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doi = "10.1021/acscatal.3c03920",

language = "English",

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journal = "ACS Catalysis",

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TY - JOUR

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

AU - Srivastava, Prabhakar L.

AU - Johns, Sam T

AU - Walters, Rebecca K

AU - Miller, David J.

AU - Van der Kamp, Marc W

AU - Allemann, Rudolf K.

N1 - Funding Information: This work was supported by the BBSRC (BB/R001596/1 and BB/R001332/1). M.W.V.d.K. was a BBSRC David Phillips Fellow (BB/M026280/1). Simulations were performed using the computational facilities of the Advanced Computing Research Centre, University of Bristol. Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.

PY - 2023/11/3

Y1 - 2023/11/3

N2 - Terpene synthases (TS) catalyse complex reactions to produce a diverse array of terpene skeletons from linear iso-prenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchou-lol. 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 sesquiter-pene synthases to produce complex cyclic hydrocarbons, without the need for structure determination or modeling.

AB - Terpene synthases (TS) catalyse complex reactions to produce a diverse array of terpene skeletons from linear iso-prenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchou-lol. 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 sesquiter-pene 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 - Letter (Academic Journal)

C2 - 37942265

SN - 2155-5435

VL - 13

SP - 14199

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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Multi-scale enzyme modelling for SynBio: optimizing biocatalysts for selective synthesis of bioactive compounds

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