Tetrahydroxynaphthalene Reductase: Catalytic Properties of an Enzyme Involved in Reductive Asymmetric Naphthol Dearomatization
Tetrahydroxynaphthalene Reductase: Catalytic Properties of an Enzyme Involved in Reductive Asymmetric Naphthol Dearomatization
复制标题
DOI：
10.1002/anie.201107695
复制
发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Mueller, Michael
中科院分区：
文献类型：
--
作者：
Schaetzle, Michael A.;Flemming, Stephan;Mueller, Michael
醇脱氢酶主要用于脂肪酮的还原。相比之下，生物合成的观点暗示了更广泛的催化活性。例如，芳香化合物在真菌和细菌中的代谢是通过好氧或厌氧途径进行的。虽然单加氧酶或双加氧酶参与好氧途径，但氧化还原酶是厌氧代谢所必需的，从而导致乙酰辅酶a的形成。此外，在次级代谢物合成过程中，多酚聚酮合成酶（PKS）产物通过还原-脱水顺序降解这种脱氧策略被认为是各种真菌1,8 -二羟基萘(DHN, 1)-黑色素生物合成的关键步骤[2-6]，也被认为是黄曲霉毒素，[7,8]放线菌素，[9]和大黄酚的形成的关键步骤。[10,11]此外，多羟基萘代表了几种次生代谢物合成中的分支点。[12 - 14]可以发现高度的代谢多样性，特别是在螺二氧萘群中DHN (1) DHN-melanin的单体的单位,是由双脱氧方式(方案1)。[2 - 6]与其产品1、3、6,8-tetrahydroxynaphthalene (T4HN 2)是减少tetrahydroxynaphthalene还原酶(T4HNR) scytalone (3), [15] scytalone容易脱水的脱水酶(SD) 1, 3, 8-trihydroxynaphthalene (T3HN, 4)。[16]以同样的方式,4是减少trihydroxynaphthalene还原酶(T3HNR) vermelone(5)和加味SD 1。[19]还原步骤被认为是通过2和4的3-酮互变异构体进行的。[20,21]参与该途径的稻瘟病病菌t4 -和T3HNR序列同源性为46%，分别表现出对T4HN(2)和T3HN(4)的偏好这两种还原酶都是短链脱氢酶/还原酶（SDR）家族的成员。这个家族以共享相同的结构基序而闻名，尽管成员表现出很少的序列相似性。[15,19,22 - 25]共同保守基序是发散性还原酶的核心结构，也是催化混杂家族的主干在天然产物合成中，脱芳化策略是一种强大而直接的循环构建模块方法。[27,28]然而，不对称催化下的一步脱芳化反应是非常具有挑战性的因此，T4HNR和T3HNR可能是催化不对称脱芳化的有价值的工具。本文将重点研究T4HNR的构效关系，以深入了解其催化循环动力学。广泛的底物范围允许鉴定萘酚底物和主要活性位点相互作用的基本结构基序。c端截断的突变效应与c端羧酸稳定的概念一致，可以解释T4HNR对底物的偏好。这种结构特征可能有助于找到进一步的萘酚还原酶。T4HNR的克隆和表达采用Thompson et al.[15]稍作修改的方法，细胞提取物无需进一步纯化即可使用。尽管其易被空气氧化，但生理底物2的转化率为47%，锡塔龙(3)的产率为33%。CD光谱测定其绝对构型为R，对映体过量（ee）为> 95%（表1及配套资料）。
Alcohol dehydrogenases are mainly applied for the reduction of aliphatic ketones. By contrast, a biosynthetic point of view hints at broader catalytic activities. For example, the metabolism of aromatic compounds in fungi and bacteria proceeds through an aerobic or anaerobic route. While mono-or dioxygenases are involved in the aerobic pathways, oxidoreductases are required for the anaerobic metabolism, thus leading to the formation of acetyl coenzyme A. Additionally, polyphenolic polyketide synthase (PKS) products degrade by a reduction–dehydration sequence during secondary metabolite synthesis.[1] This deoxygenation strategy was found as a key step in 1, 8-dihydroxynaphthalene (DHN, 1)-melanin biosynthesis of various fungi [2–6] and is also proposed for the formation of aflatoxin,[7, 8] actinorhodin,[9] and chrysophanol.[10, 11] Furthermore, polyhydroxynaphthalenes represent branching points in several secondary metabolite syntheses.[12–14] A high degree of metabolic diversity can be found, especially in the group of spirodioxynaphthalenes.[12] DHN (1), as the monomeric unit of DHN-melanin, is produced by means of a double deoxygenation (Scheme 1).[2–6] The PKS product 1, 3, 6, 8-tetrahydroxynaphthalene (T4HN, 2) is reduced by tetrahydroxynaphthalene reductase (T4HNR) to scytalone (3),[15] which is readily dehydrated by scytalone dehydratase (SD) to 1, 3, 8-trihydroxynaphthalene (T3HN, 4).[16–18] In the same manner, 4 is reduced by trihydroxynaphthalene reductase (T3HNR) to vermelone (5) and aromatized by SD to 1.[19] The reduction steps are believed to take place via the 3-keto tautomers of 2 and 4.[20, 21] T4-and T3HNR of Magnaporthe grisea involved in this route show 46% sequence identity and exhibit a preference for T4HN (2) and T3HN (4), respectively.[15] The two reductases are members of the short-chain dehydrogenase/reductase (SDR) family. This family is known for sharing the same structural motif, though the members exhibit few sequence similarities.[15, 19, 22–25] The common conserved motif is the core structure for divergent reductases, but also represents the backbone for a catalytic-promiscuous family.[26] Dearomatization strategies represent a powerful and direct approach to cyclic building blocks in natural product synthesis.[27, 28] However, dearomatization reactions concurrent with asymmetric catalysis in one step are very challenging.[27] Thus, T4HNR and T3HNR may represent valuable tools for catalytic, asymmetric dearomatization. Herein, we focus on the structure–activity relationship of T4HNR to gain insights into the dynamics of its catalytic cycle. A broad substrate range allowed for the identification of an essential structural motif of naphtholic substrates and of major active-site interactions. The mutational effect of C-terminal truncation agrees with the concept of stabilization by the C-terminal carboxylate as an explanation for the substrate preference of T4HNR. This structural feature may help to find further naphthol reductases.T4HNR was cloned and expressed using a slightly modified method of Thompson et al.[15] and the cell extract was used without further purification. Despite its susceptibility to oxidation by air, the physiological substrate 2 gave a conversion of 47% with 33% yield of scytalone (3) being obtained. The absolute configuration was determined by CD spectroscopy to be R, and the enantiomeric excess (ee) was found to be> 95%(Table 1 and the Supporting Information).