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Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase

Gonzalez Gonzalez, Veronica, Touchet, Sabrina, Grundy, Daniel J., Faraldos, Juan A. and Allemann, Rudolf Konrad 2014. Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase. Journal of the American Chemical Society 136 (41) , pp. 14505-14512. 10.1021/ja5066366

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Abstract

Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-3H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-2H6]-FDP support a germacrene–humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Chemistry
Subjects: Q Science > QD Chemistry
Publisher: American Chemical Society
ISSN: 0002-7863
Funders: BBSRC
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 2014
Last Modified: 21 Feb 2019 10:44
URI: http://orca.cf.ac.uk/id/eprint/66255

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