Calculated activation and reaction energies for the cycloaddition reactions.

<p>Calculated activation and reaction energies (B3LYP/6-311+G(d) level, kcal/mol) for the cycloaddition reactions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.g002" target="_blank">Fig. 2</a>).</p><p>Calculated activation and reaction energies for the cycloaddition reactions.</p

Computational Study of a Model System of Enzyme-Mediated [4+2] Cycloaddition Reaction

<div><p>A possible mechanistic pathway related to an enzyme-catalyzed [4+2] cycloaddition reac-tion was studied by theoretical calculations at density functional (B3LYP, O3LYP, M062X) and semiempirical levels (PM6-DH2, PM6) performed on a model system. The calculations were carried out for the key [4+2] cycloaddition step considering enzyme-catalyzed biosynthesis of Spinosyn A in a model reaction, where a reliable example of a biological Diels-Alder reaction was reported experimentally. In the present study it was demonstrated that the [4+2] cycloaddition reaction may benefit from moving along the energetically balanced reaction coordinate, which enabled the catalytic rate enhancement of the [4+2] cycloaddition pathway involving a single transition state. Modeling of such a system with coordination of three amino acids indicated a reliable decrease of activation energy by ~18.0 kcal/mol as compared to a non-catalytic transformation.</p></div

Comparison of regular and proposed cycloaddition reactions.

<p>Comparison of regular (black line) and proposed in the present study (blue line) cycloaddition reactions (calculated at the PM6 level). Coordination of amino acids and schematic substrate transformations are shown in the case of enzyme-catalyzed reaction (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.s009" target="_blank">S9 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.s010" target="_blank">S10 Fig</a> for structures and geometries).</p

Calculated activation barriers of Spinosyn A formation.

<p>Calculated ΔE^≠_2-TS activation barriers (in kcal/mol) of the cycloaddition step involved in the biosynthesis of Spinosyn A showing effect of amino acids coordination. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.s005" target="_blank">S5 Fig</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.s006" target="_blank">S6 Fig</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.s007" target="_blank">S7 Fig</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.s008" target="_blank">S8 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119984#pone.0119984.s016" target="_blank">S5 Table</a> for structures and geometric parameters.</p><p>^[a] Coordinated amino acids: 1—none; 2—Gln; 3—Gln and Ser; 4—Gln and 2 molecules of Ser.</p><p>^[b] Full geometry optimization of all stationary points.</p><p>^[c] Single point calculations at the PM6 geometry.</p><p>Calculated activation barriers of Spinosyn A formation.</p

Optimized molecular structure of transition state VIII-TS.

<p>Optimized molecular structure of transition state VIII-TS.</p

SpnF-mediated cyclization.

<p>SpnF-mediated cyclization leading to cyclohexene ring in the biosynthesis of Spinosyn A.</p

Structural decomposition analysis of the cycloaddition reaction.

<p>Structural decomposition analysis of the cycloaddition reaction involved in the biosynthesis of Spinosyn A into principal components (the atom numbering was the same as in compound <b>1</b> for comparative purpose).</p

Objects of various shapes made of PLA by 3D printing.

<p>(A) cylinder, (B) cone, (C) sphere, (D) joined cylinder/cone, (E) pyramid, (F) cube.</p

Printed cylindrical tubes of different wall thickness (l, mm).

<p>For each l value the corresponding wall structure and printing quality are displayed (all items were printed at k = 0.98).</p

Protons in the molecules of reagents and products for conversion calculation by NMR study.

<p>Protons in the molecules of reagents and products for conversion calculation by NMR study.</p