Structural and functional analysis of intermolecular Diels-Alderase

Diets-Alder反应是一种环加成反应，其机制涉及两个不饱和体系的π轨道重叠，其中烯烃（亲二烯体）与1，3-二烯加成形成6元环。该反应在温和的条件下可形成高区域和立体选择性的环状产物，因此在合成上非常有用。为了研究天然Diels-Alderase的催化机制，我们对巨噬细胞酸合成酶（MPS）的晶体结构进行了分析。首先，我们给出了这种多功能酶的X-射线晶体结构。这是天然Diels-Alderase的第一个原子分辨率结构。活性位点揭示了催化Diets-Alder反应的几个意想不到的特征，包括与2-吡喃酮形成催化重要氢键的残基的存在。2-吡喃酮C3和C5位上的吸电子基团增加了二烯的反应活性，此外，刘易斯酸的配位或活性中心溶剂和催化剂残基与羰基的氢键作用有效地降低了二烯的LUMO能，导致反应活性的增加。我们可以看到天然酶与人工催化抗体相比的优势;由这些酶催化的第一反应的产物被捕获在活性位点中，与[4+2]反应的其他部分的π轨道重叠并形成两个新的碳-碳键。这类天然Diels-Alderases具有利用在反应介质中不稳定的高反应性底物的明显优势。

Diets-Alder reaction is a cycloaddition whose mechanism involves the overlap of the pi-orbitals of the two unsaturated systems in which an alkene (dienophile) adds to a 1,3-diene to form a 6-membered ring. The reaction is synthetically very useful because it forms cyclic products with high regio-and stereoselectivity under mild conditions. To investigate the catalytic mechanism of the natural Diels-Alderase, we initiated the crystal structure analysis of macrophomate synthase (MPS). Firstly we presented the X-ray crystal structure of this multifunctional enzyme. This is the first atomic resolution structure of the natural Diels-Alderase. The active site reveals several unexpected features for catalyzing the Diets-Alder reaction, including the existence of the residues for making catalytically important hydrogen bonds to the 2-pyrone. The electron-withdrawing groups at C3 and C5 of the 2-pyrone increase the reactivity of diene and moreover, the coordination of Lewis acid or the hydrogen bonding of solvent and catalytic residues in active site with the carbonyl groups effectively decreases the LUMO energy of the diene, resulting in an increase of the reactivity. We can see the advantage of natural enzymes compared to artificial catalytic antibodies ; the products of a first reaction catalyzed by these enzymes are trapped in an active site, overlapping of pi-orbitals with other moiety for a [4+2] reaction and forming two new carbon-carbon bonds. This type of natural Diels-Alderases has obvious advantage to utilize highly reactive substrate that is not stable in reaction medium.