Biosynthesis of mycarose: Isolation and characterization of enzymes involved in the C-2 deoxygenation
Biosynthesis of mycarose: Isolation and characterization of enzymes involved in the C-2 deoxygenation
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DOI：
10.1021/ja991713o
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发表时间：
1999-09-08
影响因子：
15
通讯作者：
Liu, HW
中科院分区：
文献类型：
--
作者：
Chen, HW;Agnihotri, G;Liu, HW
碳氧键的断裂是一个非常重要的反应，对于从DNA合成到能量代谢的许多生理过程都是必不可少的。因此，进化出各种各样的机制来促进生物CO键断裂并不奇怪。1这种机制的一个更有趣的例子涉及在蛔虫素生物合成过程中基于自由基的3-脱氧步骤。2，3在该系统中，CDP-4-酮-6-脱氧-D-葡萄糖的3-OH通过吡哆胺5′-磷酸（PMP）依赖性含[2Fe-2S]的酶（E1）4和NADH依赖性还原酶（E3）之间的协同催化作用不可逆地切除。5由于常见糖的脱氧似乎是自然界采用的一种看似简单但有效的修饰，以产生碳水化合物结构排列，因此脱氧糖生物合成的研究提供了一个很好的机会来了解自然界打破不同CO键的各种策略。1由于新的途径和与C-3脱氧相关的酶活性的成功表征，2，3目前越来越多的注意力转移到参与2，6-二脱氧己糖生物合成的C-2脱氧事件，其通常存在于次级代谢产物中。图2b，c对于真菌糖（2）来说无疑是这样，真菌糖是一种具有C-3甲基分支的2，6-双脱氧糖，是大环内酯类抗生素泰乐菌素（1）的三种糖组分之一，由草莓链霉菌产生。早期的遗传学研究产生了负责泰乐菌素生物合成的整个基因簇（方案1）6，并且还揭示了参与真菌糖生物合成的基因位于tylCK和tylIBA区域。最近，我们对这两个区域进行了测序，从中鉴定了12个开放阅读框架（ORF）。7，8如方案1所示，根据与其它糖生物合成基因，特别是来自红霉素生物合成基因簇的那些基因的序列相似性，确定了被认为参与真菌糖形成的基因，9，其还含有真菌糖衍生物虽然在真菌糖途径中2-OH的去除可能遵循类似于由E1和E3催化的C-3脱氧的机制，图2b，c中，泰乐菌素和红霉素簇中同源E1和E3基因的缺失导致了另一种脱水机制（参见方案1）。在对含有1，4-连接至苯并异色满醌发色团的2，6-二脱氧-D-己糖部分的石榴苷和石榴苷B的生物合成的研究中，Draeger等人报道了能够将3转化为稳定的TDP-4-酮-2，6-二脱氧葡萄糖产物的脱氢酶（Gra Orf 27）和还原酶（Gra Orf 26）的鉴定。[11]他们的数据提供了第一个生化证据，证实了所提出的脱水机制。在这里，我们报告了我们自己对真菌糖生物合成中C-2脱氧机制的研究结果（2）。我们的结果不仅证实了Draeger等人的结论，但更重要的是，我们对参与C-2脱氧的酶的生物化学表征也提供了对其催化模式的重要见解。如方案1所示，tylIBA区域下游的tylX 3基因被指定为负责C-2脱氧事件的还原酶，而tylCK区域内的tylC 1被推测为还原酶基因。为了获得TylX 3和TylC 1蛋白，通过聚合酶链式反应（PCR）扩增它们各自的编码区。克隆了tylX 3基因...
The breaking of carbon-oxygen bonds is a fundamentally important reaction, essential for many physiological processes ranging from DNA synthesis to energy metabolism. It is thus not surprising that a diverse array of mechanisms have evolved to facilitate biological CO bond scission. 1 One of the more interesting examples of such a mechanism involves the radical-based 3-deoxygenation step during ascarylose biosynthesis. 2, 3 In this system, the 3-OH of CDP-4-keto-6-deoxy-D-glucose is irreversibly excised by a collaborative catalysis between a pyridoxamine 5′-phosphate (PMP)-dependent [2Fe-2S]-containing enzyme (E1) 4 and a NADH-dependent reductase (E3). 5 Since deoxygenation of common sugars appears to be a seemingly simple yet effective modification adopted by nature to generate carbohydrate structural permutations, the study of deoxysugar biosynthesis offers an excellent opportunity to learn nature’s diverse strategies for breaking different CO bonds. 1 Due to the successful characterization of the novel pathway and enzymatic activities associated with C-3 deoxygenation, 2, 3 increasing attention is presently being shifted to the C-2 deoxygenation event involved in the biosynthesis of 2, 6-dideoxyhexoses, which are commonly found in secondary metabolites. 2b, c This is certainly the case for mycarose (2), a 2, 6-dideoxy sugar with a C-3 methyl branch that is one of the three sugar components of the macrolide antibiotic tylosin (1), produced by Streptomyces fradiae. Early genetic studies yielded the entire gene cluster responsible for the biosynthesis of tylosin (Scheme 1) 6 and also revealed that the genes involved in the biosynthesis of mycarose are in the tylCK and tylIBA regions. Recently, we have sequenced these two regions, from which 12 open reading frames (ORFs) were identified. 7, 8 As shown in Scheme 1, the genes believed to participate in the formation of mycarose are assigned on the basis of sequence similarities to other sugar biosynthetic genes, particularly those derived from the biosynthetic gene cluster of erythromycin, 9 which also contains a mycarose derivative (cladinose) as one of its appended sugars.While removal of the 2-OH in the mycarose pathway may follow a mechanism analogous to that for C-3 deoxygenation catalyzed by E1 and E3, 2b, c the absence of the homologous E1 and E3 genes in the tylosin and erythromycin clusters has led to an alternative dehydration mechanism (see Scheme 1). 9, 10 In a study of the biosynthesis of granaticin and granaticin B, which contain a 2, 6-dideoxy-D-hexose moiety 1, 4-linked to a benzoisochromane quinone chromophore, Draeger et al. reported the identification of a dehydratase (Gra Orf 27) and a reductase (Gra Orf26) that could convert 3 to a stable TDP-4-keto-2, 6-dideoxyglucose product. 11 Their data provided the first biochemical evidence validating the proposed dehydration mechanism. Here, we report the findings of our own investigations into the mechanism of C-2 deoxygenation in the biosynthesis of mycarose (2). Not only do our results corroborate the conclusion reached by Draeger et al., but more importantly, our biochemical characterization of the enzymes involved in C-2 deoxygenation also offers significant insights into the mode of their catalyses. As depicted in Scheme 1, the tylX3 gene downstream of the tylIBA region has been assigned as the dehydratase responsible for the C-2 deoxygenation event, while tylC1 within the tylCK region has been speculated to be the reductase gene. To obtain the TylX3 and TylC1 proteins, their respective coding regions were amplified by the polymerase chain reaction (PCR). The tylX3 gene was cloned …