Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.

Veronica Gonzalez,Sabrina Touchet,Daniel J. Grundy,Juan A. Faraldos,Rudolf K. Allemann

doi:10.1021/ja5066366

Veronica Gonzalez, Sabrina Touchet + Show 3 more

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https://doi.org/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-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-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.

Highlights

Class I sesquiterpene synthases catalyze the conversion of the linear substrate (2E,6E)-farnesyl diphosphate (1, FDP) to all C15 isoprenoid hydrocarbons found in nature.[1]
Farnesyl cation (2) loses a proton to yield linear farnesene hydrocarbons[3] or reacts with another double bond in 2 to generate an often tertiary carbocation that is chaperoned by the enzyme toward the product through a well-defined energetic landscape and with extraordinary regio- and stereochemical precision.[4]
10.0 ± 0.9 7.3 ± 0.2 7.9 ± 0.1 2.5 ± 0.2 6.2 ± 0.3 0.5 ± 0.03 bility, we investigated the products generated by Germacrene A synthase (GAS) and some mutants

Summary

■ INTRODUCTION

Class I sesquiterpene synthases catalyze the conversion of the linear substrate (2E,6E)-farnesyl diphosphate (1, FDP) to all C15 isoprenoid hydrocarbons found in nature.[1]. Phylogenetic analyses of plant sesquiterpene synthases[9] together with crystallographic data from structural work1c and structural models have led to the suggestion that primordial plant sesquiterpene synthases might have performed only 1,11- and 1,6-cyclizations, while synthases that catalyze 1,10-cyclization pathways have evolved more recently via gene duplication and subsequent mutations.[10] it might be possible to reconstruct a 1,11-cyclization enzyme from a modern 1,10-sesquiterpene synthase. This mutant GAS might represent an example of a 1,11-ancestor of modern plant 1,10-synthases

■ RESULTS AND DISCUSSION

■ ACKNOWLEDGMENTS

■ REFERENCES