Structural and functional analysis of intermolecular Diels-Alderase

Diets-Alder反应是一种环加成反应，其机理涉及两个不饱和体系的pi-轨道重叠，其中烯烃（亲二烯）与1,3-二烯加在一起形成一个6元环。该反应在温和条件下生成具有高区域选择性和立体选择性的环产物，在合成上非常有用。为了研究天然Diels-Alderase的催化机制，我们对巨磷酸合成酶（MPS）的晶体结构进行了分析。首先给出了该多功能酶的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-吡酮形成催化重要氢键的残基的存在。2-吡酮的C3和C5上的吸电子基团提高了二烯的反应活性，而活性位点的溶剂和催化残基与羰基的Lewis酸配位或氢键有效降低了二烯的LUMO能，从而提高了反应活性。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困在一个活性位点上，与[4+2]反应的其他部分重叠，形成两个新的碳-碳键。这种天然diels - alderase具有明显的优势，可以利用在反应介质中不稳定的高活性底物。

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.