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首页> 外文期刊>Journal of Molecular Biology >Crystal structure and catalytic mechanism of leucoanthocyanidin reductase from Vitis vinifera.
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Crystal structure and catalytic mechanism of leucoanthocyanidin reductase from Vitis vinifera.

机译：葡萄中白花色素苷还原酶的晶体结构和催化机理。

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摘要

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.
机译：白花青素还原酶（LAR）催化NADPH依赖性的2R，3S，4S-flavan-3,4-二醇还原为2R，3S-flavan-3-ols，这是类黄酮的亚家族，对植物存活和人类营养至关重要。来自葡萄的LAR1与或不与NADPH及其天然产物之一（+）-儿茶素共结晶。晶体衍射至1.75和2.72 A之间的分辨率。辅酶和底物结合袋在脱辅基蛋白中预先形成，在NADPH结合后不会发生明显变化。以2.28 A的分辨率确定的流产三元复合物的结构表明与底物结合相关的短3（10）螺旋的顺序，并表明His122和Lys140充当酸碱催化剂。基于我们的3D结构，提出了一个两步催化机制，其中协同脱水发生在NADPH介导的氢化物在C4处转移。脱水步骤包括通过桥连的水分子对酚式OH7进行Lys催化的去质子化，以及在C4处对苄基羟基的His催化的质子化。所得的醌甲基化物用作C 4处氢化物转移的亲电子目标。 LAR属于短链脱氢酶/还原酶超家族，并且属于PIP（松脂醇-lariciresinol还原酶，异黄酮还原酶和苯基香豆素苄基醚还原酶）家族。我们的数据支持以下概念：所有PIP酶均会还原醌甲基化物中间体，并且短链脱氢酶/还原酶催化三联体（Ser ... TyrXXXLys）保留的唯一残基（赖氨酸，通过催化酚羟基的去质子化来促进该中间体的形成。对于某些PIP酶来说，赖氨酸催化的质子提取可能足以触发离去基团的挤出，而在LAR中，氢氧化物基团的挤出需要更复杂的酸化催化协同机制，该机制涉及组氨酸并利用白花色素的OH 4，OH 5和OH 7取代基中的一个。

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