Menaquinone biosynthesis in Escherichia coli:: Identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-l-carboxylate as a novel intermediate and re-evaluation of MenD activity
Menaquinone biosynthesis in Escherichia coli:: Identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-l-carboxylate as a novel intermediate and re-evaluation of MenD activity
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DOI：
10.1021/bi700810x
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发表时间：
2007-09-25
期刊：
影响因子：
2.9
通讯作者：
Guo, Zhihong
中科院分区：
文献类型：
--
作者：
Jiang, Ming;Cao, Yang;Guo, Zhihong
甲基萘醌是大肠杆菌厌氧生长期间呼吸链中的电子载体。其生物合成涉及(1R,6R)-2-琥珀酰-6-羟基-2,4-环己二烯-1-羧酸(SCHHC)作为中间体，其被认为是由生物合成酶之一MenD从异分支酸和2-酮戊二酸衍生而来。然而，我们发现真正的 MenD 产品是 2-琥珀酰-5-烯醇丙酮酰-6-羟基-3-环己烯-1-羧酸 (SEPCHC)，而不是 SCHHC。以下发现支持这一点：(i) 异分支酸消耗和 SCHHC 形成在酶反应中不同步，(ii) SCHHC 形成速率与酶浓度无关，(iii) SCHHC 在异分支酸底物很容易被 MenD 消耗的弱酸性或中性溶液中不形成，以及 (iv) 在禁止 SCHHC 形成的条件下形成的 MenD 周转产物具有与 SEPCHC 结构一致的光谱特征，在弱碱性溶液中自发进行 2,5-消除反应，形成 SCHHC 和丙酮酸。该中间体的两个特性，即紫外线透明度和化学稳定性，为 SCHHC 一直被误认为是 MenD 产品提供了理由。根据这些发现，MenD 被重新发现是一种具有高二阶速率常数的高效酶，应更名为 SEPCHC 合酶。有趣的是，负责将 SEPCHC 转化为 SCHHC 的酶活性似乎与甲基萘醌生物合成中的任何已知酶无关，但存在于大肠杆菌 K12 的粗提物中，这表明真正的 SCHHC 合酶仍有待鉴定，以充分阐明普遍存在的生物合成途径。
Menaquinone is an electron carrier in the respiratory chain of Escherichia coli during anaerobic growth. Its biosynthesis involves (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) as an intermediate, which is believed to be derived from isochorismate and 2-ketoglutarate by one of the biosynthetic enzymes-MenD. However, we found that the genuine MenD product is 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC), rather than SHCHC. This is supported by the following findings: (i) isochorismate consumption and SHCHC formation are not synchronized in the enzymic reaction, (ii) the rate of SHCHC formation is independent of the enzyme concentration, (iii) SHCHC is not formed in weakly acidic or neutral solutions in which the isochorismate substrate is readily consumed by MenD, and (iv) the MenD turnover product, formed under conditions disabling SHCHC formation, possesses spectroscopic characteristics consistent with the structure of SEPHCHC and spontaneously undergoes 2,5-elimination to form SHCHC and pyruvate in weakly basic solutions. Two properties of the intermediate, ultraviolet transparency and chemical instability, provide a rationale for the fact that SHCHC has been consistently mistaken as the MenD product. In accordance with these findings, MenD was rediscovered to be a highly efficient enzyme with a high second-order rate constant and should be renamed SEPHCHC synthase. Intriguingly, the enzymatic activity responsible for conversion of SEPHCHC into SHCHC appears not to associate with any of the known enzymes in menaquinone biosynthesis but is present in the crude extract of E. coli K12, suggesting that a genuine SHCHC synthase remains to be identified to fully elucidate the ubiquitous biosynthetic pathway.