9 research outputs found
Alternative Synthesis of the Colorado Potato Beetle Pheromone
A concise
preparation of the pheromone secreted by the male Colorado
potato beetle [viz. (3<i>S</i>)-1,3-dihydroxy-3,7-dimethyl-6-octen-2-one]
was accomplished in four steps starting from 2-fluoronerol or 2-fluorogeraniol.
The key step in the synthesis involves a 6-endo epoxide ring-opening
with ester participation that simultaneously inverts the 3<i>R</i>-configuration of the (3<i>R</i>)-2,3-epoxy-2-fluoroprenyl
acetate intermediate and installs the ketone functionality of the
semiochemical. Extensive NMR studies validate the proposed 6-endo
mechanism of the featured rearrangement, which under anhydrous conditions
resulted in the formation of two bicyclic 1,3-dioxan-5-ones via an
unprecedented intramolecular Prins cyclization
Evolutionary and Mechanistic Insights from the Reconstruction of Ī±āHumulene Synthases from a Modern (+)-Germacrene A Synthase
Germacrene
A synthase (GAS) from <i>Solidago canadensis</i> 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% Ī±-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-<sup>3</sup>H<sub>1</sub>]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments
with [12,13-<sup>2</sup>H<sub>6</sub>]-FDP support a germacreneāhumulene
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
Probing the Mechanism of 1,4-Conjugate Elimination Reactions Catalyzed by Terpene Synthases
The reaction mechanisms of (<i>E</i>)-Ī²-farnesene
synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze
a formal regiospecific 1,4-conjugate elimination of hydrogen diphosphate
from (<i>E</i>,<i>E</i>)-farnesyl and dimethylallyl
diphosphate (FDP and DMADP) to generate the semiochemicals (<i>E</i>)-Ī²-farnesene and isoprene, respectively, were probed
with substrate analogs and kinetic measurements. The results support
stepwise reaction mechanisms through analogous enzyme-bound allylic
cationic intermediates. For EBFS, we demonstrate that the elimination
reaction can proceed via the enzyme-bound intermediate <i>trans</i>-nerolidyl diphosphate, while for ISPS the intermediacy of 2-methylbut-3-enyl
2-diphosphate can be inferred from the product outcome when deuterated
DMADPs are used as substrates. Possible implications derived from
the mechanistic details of the EBFS-catalyzed reaction for the evolution
of sesquiterpene synthases are discussed
Growth behaviour of <i>P. capsici</i> tdtom, after 10 days of exposure to different capsidiol concentrations.
<p>The experiment was performed 3 times.</p
Scatter plots correlating 0D600 and capsidiol concentration.
<p>The plots illustrate growth of <i>P. infestans</i> 88069td (A) and <i>P. capsici</i> tdtom (B) strains over time for a maximum of 10 days. The experiment was performed 3 times.</p
Scatter plots correlating fluorescence intensity and capsidiol concentration.
<p>The plots illustrate fluorescence intensity of <i>P. infestans</i> 88069td (A) and <i>P. capsici</i> tdtom (B) strains over time for a maximum of 10 days. The experiment was performed 3 times.</p
Capsidiol inhibits <i>P. infestans</i> growth reversibly.
<p>(A) Growth inhibition assay of <i>P. infestans</i> after 10 days of exposure of mycelial plugs to capsidiol. (B) Restoration of growth after washing treatment. Green line indicates the point after which the washing treatment was applied. The experiment was performed 3 times. Picture was taken 10 days after the washing and 20 days after initial exposure to capsidiol.</p
Structural Elucidation of Cisoid and Transoid Cyclization Pathways of a Sesquiterpene Synthase Using 2-Fluorofarnesyl Diphosphates
Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (<i>trans</i>,<i>trans</i>)-farnesyl diphosphate (FPP) (<b>1a</b>) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) to exclusively (ā„99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-<i>trans</i> FPP (<b>1a</b>) and (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed