Electrostatically Guided Dynamics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 (^PhImC) 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_em 398–420 nm, Φ 0.009–0.687), and excellent correlation was found between Φ of <b>5a</b>–<b>g</b> and σ_p^– of the substituent on the phenyl-imidazolo moiety (<i>R</i>² = 0.94). Calculations suggested that the dominant tautomer of ^PhImC in methanol solution is identical to that of cytidine. DFT calculations of the stable tautomer of selected ^PhImC analogues suggested a relationship between the HOMO–LUMO gap and Φ and explained the loss of fluorescence in the nitro analogue. Incorporation of the CF₃-^PhImdC 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>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><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>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b>:<b>ODN­(G)</b> was reduced 17-fold vs that of a single strand, whereas that of <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b>:<b>ORN­(G)</b> was reduced only 3.8-fold, <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b> appears to be a DNA-selective probe. We conclude that the <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><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_em) of <b>5</b>–<b>17</b> or their quantum yields (φ), and Hammett constants (σ<i>_p</i> and σ<i>_m</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_em 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_abs 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 IC₅₀ 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 0.5x10⁶ 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). IC₅₀ 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