Chimie et Biologie des Membranes et des Nanoobjets, UMR CNRS 5248, Bât. B8, Avenue des Facultés, Université Bordeaux 1, 33405 Talence Cedex, France.
J Mol Biol. 2010 Apr 9;397(4):1079-91. doi: 10.1016/j.jmb.2010.02.002. Epub 2010 Feb 6.
Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 A. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 A, indicates the ordering of a short 3(10) helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser...TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.
类查耳酮还原酶（LAR）催化 NADPH 依赖性的 2R,3S,4S-黄烷-3,4-二醇还原为 2R,3S-黄烷-3-醇，该物质是类黄酮的一个亚家族，对植物的生存和人类的营养都很重要。葡萄的 LAR1 已与 NADPH 共结晶，或不与 NADPH 共结晶，以及与其天然产物之一 (+)-儿茶素共结晶。晶体的分辨率在 1.75 到 2.72 A 之间。辅酶和底物结合口袋在脱辅基蛋白中预先形成，并且在与 NADPH 结合时不会明显改变。在 2.28 A 的分辨率下确定的无效三元复合物的结构表明，与底物结合相关的短 3(10)螺旋的有序排列，并表明 His122 和 Lys140 起酸碱催化剂的作用。基于我们的 3D 结构，提出了一个两步催化机制，其中协同脱水先于 C4 处的 NADPH 介导的氢转移。脱水步骤涉及 Lys 催化的通过桥水分子去质子化酚 OH7，以及 His 催化的 C4 位苄醇羟基的质子化。所得的醌甲亚胺作为 C4 处氢转移的亲电靶标。LAR 属于短链脱氢酶/还原酶超家族和 PIP（松脂醇-二氢马桑素还原酶、异黄酮还原酶和苯并呋喃香豆素苄醚还原酶）家族。我们的数据支持所有 PIP 酶还原醌甲亚胺中间体的概念，并且短链脱氢酶/还原酶催化三联体（Ser...TyrXXXLys）中唯一保守的残基（赖氨酸）的主要作用是通过催化酚羟基的去质子化来促进该中间体的形成。对于一些 PIP 酶，这种赖氨酸催化的质子提取可能足以引发离去基团的排出，而在 LAR 中，氢氧根的排出需要更复杂的协同酸碱催化机制，该机制涉及组氨酸并利用查耳酮的 OH4、OH5 和 OH7 取代基。
