8 research outputs found
Electrostatically Guided Dynamicsî—¸The Root of Fidelity in a Promiscuous Terpene Synthase?
Terpene cyclases are responsible for the initial cyclization
cascade
in the multistep synthesis of more than 60 000 known natural
products. This abundance of compounds is generated using a very limited
pool of substrates based on linear isoprenoids. The astounding chemodiversity
obtained by terpene cyclases suggests a tremendous catalytic challenge
to these often promiscuous enzymes. In the current study we present
a detailed mechanistic view of the biosynthesis of the monoterpene
bornyl diphosphate (BPP) from geranyl diphosphate by BPP synthase
using state of the art simulation methods. We identify the bornyl
cation as an enzyme-induced bifurcation point on the multidimensional
free energy surface, connecting between the product BPP and the side
product camphene. Chemical dynamics simulations suggest that the active
site diphosphate moiety steers reaction trajectories toward product
formation. Nonetheless, chemical dynamics is not precise enough for
exclusive product formation, providing a rationale for the lack of
fidelity in this promiscuous terpene cyclase
Phenyl-imidazolo-cytidine Analogues: Structure–Photophysical Activity Relationship and Ability To Detect Single DNA Mismatch
To expand the arsenal of fluorescent
cytidine analogues for the
detection of genetic material, we synthesized <i>para</i>-substituted phenyl-imidazolo-cytidine (<sup>Ph</sup>ImC) analogues <b>5a</b>–<b>g</b> and established a relationship between
their structure and fluorescence properties. These analogues were
more emissive than cytidine (λ<sub>em</sub> 398–420 nm,
Φ 0.009–0.687), and excellent correlation was found between
Φ of <b>5a</b>–<b>g</b> and σ<sub>p</sub><sup>–</sup> of the substituent on the phenyl-imidazolo moiety
(<i>R</i><sup>2</sup> = 0.94). Calculations suggested that
the dominant tautomer of <sup>Ph</sup>ImC in methanol solution is
identical to that of cytidine. DFT calculations of the stable tautomer
of selected <sup>Ph</sup>ImC analogues suggested a relationship between
the HOMO–LUMO gap and Φ and explained the loss of fluorescence
in the nitro analogue. Incorporation of the CF<sub>3</sub>-<sup>Ph</sup>ImdC analogue into a DNA probe resulted in 6-fold fluorescence quenching
of the former. A <i>17-fold</i> reduction of fluorescence
was observed for the <i>G-matched</i> duplex vs <b>ODNÂ(CF</b><sub><b>3</b></sub><b>-</b><sup><b>Ph</b></sup><b>ImdC)</b>, while for <i>A-mismatched</i> duplex,
only a <i>2-fold</i> decrease was observed. Furthermore,
since the quantum yield of <b>ODNÂ(CF</b><sub><b>3</b></sub><b>-</b><sup><b>Ph</b></sup><b>ImdC)</b>:<b>ODNÂ(G)</b> was reduced 17-fold vs that of a single strand, whereas
that of <b>ODNÂ(CF</b><sub><b>3</b></sub><b>-</b><sup><b>Ph</b></sup><b>ImdC)</b>:<b>ORNÂ(G)</b> was
reduced only 3.8-fold, <b>ODNÂ(CF</b><sub><b>3</b></sub><b>-</b><sup><b>Ph</b></sup><b>ImdC)</b> appears
to be a DNA-selective probe. We conclude that the <b>ODNÂ(CF</b><sub><b>3</b></sub><b>-</b><sup><b>Ph</b></sup><b>ImdC)</b> probe, exhibiting emission sensitivity upon single
nucleotide replacement, may be potentially useful for DNA single nucleotide
polymorphism (SNP) typing
Rules for the Design of Highly Fluorescent Nucleoside Probes: 8‑(Substituted Cinnamyl)-Adenosine Analogues
Currently, there are no tools that
can help the design of useful
fluorescent analogues. Hence, we synthesized a series of 8-(substituted
cinnamyl)-adenosine analogues, <b>5</b>–<b>17</b>, and established a relationship between their structure and fluorescence
properties. We attempted to find a correlation between maximum emission
wavelengths (λ<sub>em</sub>) of <b>5</b>–<b>17</b> or their quantum yields (φ), and Hammett constants
(σ<i><sub>p</sub></i> and σ<i><sub>m</sub></i>) of the substituent on the cinnamyl moiety. A linear correlation
was observed at low-medium σ values, but not at high σ
values (≥0.7). Next, we explored correlation between λ<sub>em</sub> and φ of <b>5</b>–<b>17</b> and
computed HOMO and LUMO energy levels of fragments of <b>5</b>–<b>17</b>, i.e., 8-vinyl 9-Me-adenine (fluorescent
molecule), <b>18</b>, and substituted toluene rings (fluoresence
modulators), <b>19</b>–<b>30</b>. High φ
correlated with relatively close LUMO levels of <b>19</b>–<b>30</b> and <b>18</b> (−0.076 to −0.003 eV).
The electron density of LUMO of nitro analogues <b>9</b> and <b>15</b> is localized on the aryl ring only, which explains their
low φ. Calculation of HOMO–LUMO gap of <b>5</b>–<b>17</b> enables accurate prediction of the λ<sub>abs</sub> for a planned analogue, and LUMO levels of an aryl moiety
vs 8-vinyl 9-Me-adenine, allows the prediction of high or low φ.
These findings lay the ground for prediction of fluorescence properties
of additional analogues having a similar structure
Chemical Control in the Battle against Fidelity in Promiscuous Natural Product Biosynthesis: The Case of Trichodiene Synthase
Terpene cyclases catalyze the highly
stereospecific molding of polyisoprenes into terpenes, which are precursors
to most known natural compounds. The isoprenoids are formed via intricate
chemical cascades employing rich, yet highly erratic, carbocation
chemistry. It is currently not well understood how these biocatalysts
achieve chemical control. Here, we illustrate the catalytic control
exerted by trichodiene synthase, and in particular, we discover two
features that could be general catalytic tools adopted by other terpenoid
cyclases. First, to avoid formation of byproducts, the enzyme raises the energy
of bisabolyl carbocation, which is a general mechanistic branching
point in many sesquiterpene cyclases, resulting in an essentially
concerted cyclization cascade. Second, we identify a sulfur–carbocation
dative bonding interaction that anchors the bisabolyl cation in a reactive conformation,
avoiding tumbling and premature deprotonation. Specifically, Met73
acts as a chameleon, shifting from an initial sulfur−π
interaction in the Michaelis complex to a sulfur–carbocation
complex during catalysis
Effect of SAHA and AN-7 on the viability of MF/SS cell lines, SPBL and NPBL.
<p>Viability curves based on the MTT assay of MyLa cells, Hut78 cells, (a,b), and SPBL (n = 3) (c,d) compared to NPBL (n = 8) following treatment with SAHA (a,c) and AN-7 (b,d) for 72 h. Also shown are the IC<sub>50</sub> and SI values of SAHA and AN-7 in MF/SS cell lines and SPBL and NPBL based on viability curves a-d, and their p values (e).</p
Apoptosis induction of AN-7 and SAHA in SPBL.
<p>PBL from 2 SS patients were plated at a concentration of 0.5x10<sup>6</sup> cells/mL, and were then treated with SAHA 4 μM or AN-7 200 μM for 48 h. The cells were then stained with annexin V and PI. FACS plots are shown with percent of cells in each quadruplet, and the percent of cells in apoptotic cells (early + late apoptosis) are shown also in column.</p
Toxic and apoptotic effect of SAHA and AN-7 on MF/SS cell lines as a function of exposure time.
<p>Viability curves based on trypan blue staining of MyLa and Hut78 cells following short or long exposure to SAHA (a, c) or AN-7 (b, d). IC<sub>50</sub> values of short and long exposure to SAHA and AN-7 in MF/SS cell lines based on viability curves a-d (e). Apoptosis curves based on FACS analysis of annexin V and PI staining (f-i). Percent of apoptotic MyLa cells (early + late apoptosis) after short or long exposure to SAHA (f) or AN-7 (g), and apoptotic Hut78 cells after short or continuous exposure to SAHA (h) or AN-7 (i).</p
Effect of SAHA and AN-7 on specific protein expression and modification in MF/SS cell lines.
<p>Immunoblot of apoptotic and proapoptotic proteins in MyLa and Hut78 cells treated with SAHA 10 μM or AN-7 300 μM for the indicated periods (a). Basal HDAC1 protein expression in NPBL and MF/SS cell lines (b) and in MF/SS cell lines treated with SAHA 10 μM or AN-7 300 μM (c). Acetylated H3 in the nuclear lysate of MF/SS cell lines treated with and the same concentrations of SAHA and AN-7 for the indicated periods (d).</p