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Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

[Image: see text] The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ide...

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Autores principales: Li, Ruiqiong, Chou, Wayne K. W., Himmelberger, Julie A., Litwin, Kevin M., Harris, Golda G., Cane, David E., Christianson, David W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985761/
https://www.ncbi.nlm.nih.gov/pubmed/24517311
http://dx.doi.org/10.1021/bi401643u
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author Li, Ruiqiong
Chou, Wayne K. W.
Himmelberger, Julie A.
Litwin, Kevin M.
Harris, Golda G.
Cane, David E.
Christianson, David W.
author_facet Li, Ruiqiong
Chou, Wayne K. W.
Himmelberger, Julie A.
Litwin, Kevin M.
Harris, Golda G.
Cane, David E.
Christianson, David W.
author_sort Li, Ruiqiong
collection PubMed
description [Image: see text] The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.
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spelling pubmed-39857612015-02-11 Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase Li, Ruiqiong Chou, Wayne K. W. Himmelberger, Julie A. Litwin, Kevin M. Harris, Golda G. Cane, David E. Christianson, David W. Biochemistry [Image: see text] The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. American Chemical Society 2014-02-11 2014-02-25 /pmc/articles/PMC3985761/ /pubmed/24517311 http://dx.doi.org/10.1021/bi401643u Text en Copyright © 2014 American Chemical Society
spellingShingle Li, Ruiqiong
Chou, Wayne K. W.
Himmelberger, Julie A.
Litwin, Kevin M.
Harris, Golda G.
Cane, David E.
Christianson, David W.
Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase
title Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase
title_full Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase
title_fullStr Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase
title_full_unstemmed Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase
title_short Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase
title_sort reprogramming the chemodiversity of terpenoid cyclization by remolding the active site contour of epi-isozizaene synthase
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985761/
https://www.ncbi.nlm.nih.gov/pubmed/24517311
http://dx.doi.org/10.1021/bi401643u
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