3′-Enolpyruvyl-UMP, a novel and unexpected metabolite in nikkomycin biosynthesis
3′-Enolpyruvyl-UMP, a novel and unexpected metabolite in nikkomycin biosynthesis
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DOI：
10.1002/cbic.200500208
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发表时间：
2005-11-01
期刊：
影响因子：
3.2
通讯作者：
Macheroux, P
中科院分区：
文献类型：
--
作者：
Ginj, C;Rüegger, H;Macheroux, P
尼可霉素是由几种链霉菌产生的，由于它们对几丁质合成酶有很强的抑制作用，因此具有杀菌、杀虫和杀螨性。[1-4]从结构上讲，它们可以归类为含有两种不常见氨基酸的肽基核苷，即羟基吡啶高苏氨酸和具有N-糖基连接碱基的氨基己糖酸(方案1)。[5]虽然尼可霉素的化学结构早在20世纪70年代就已经知道，但关于它们的生物合成的信息很少。在克隆了与尼克霉素生物合成有关的一整套结构基因之后，[6]对合成4-甲酰基-4-咪唑啉-2-酮碱的酶促步骤进行了较详细的研究。[7-9]多肽部分是由11个酶反应合成的，其中只有两个得到了深入的研究。[10，11]氨基己糖醛酸是通过将5-磷酸核糖-1-焦磷酸转移到碱基(尿嘧啶或4-甲酰基-4-咪唑啉-2-酮)上，然后再加成一个烯丙酮基来进入尼克霉素骨架的。推测为核糖的5‘-羟基。[12，13]这种假定的中间体随后被相当推测的反应进一步修饰，以产生氨基己糖酸前体。[13]根据氨基酸序列相似性，尼可霉素操纵子中的Niko基因似乎编码一种烯醇式丙酮酸转移酶。[13]这个酶家族由两种特性良好的酶组成，即5-烯醇式丙酮酸-莽草酸3-磷酸合成酶(EPSPS，EC 2.5)。1.19)和UDP-N-乙酰氨基葡萄糖烯醇式丙酮酸转移酶(Mura，EC 2.5.1.7)，催化完整的烯醇丙酮基从PEP转移到莽草酸3-磷酸的5-羟基和UDP-N-乙酰氨基葡萄糖的3‘-羟基。因此，Niko有望催化尼克霉素生物合成中烯醇式丙酮基转移反应。为了证实Niko的作用，我们克隆了肌腱链霉菌Tü901的基因，并在大肠杆菌中进行了异源表达。重组蛋白被纯化为均一，并对其活性进行了分析。[14]与预期相反，以尿苷为底物时没有发现烯醇式丙酮基转移酶的活性。酶分析是通过比色终点法和利用嘌呤核苷磷酸化酶反应的连续分光光度酶偶联方法测量释放的磷酸盐来执行的。[14-16]这些分析中观察到的磷酸盐释放伴随着由UMP和PEP生成的新的核苷酸化合物，如高效液相分析所证明的那样。[14]对反应产物的分析表明，在酶反应过程中，随着新化合物的形成，磷酸盐以化学计量的方式释放，这表明核苷酸中仍然有一个磷酸基团。用~(32)P标记的UMP在酶反应中证实了这一结果：保留了90%的放射性标记(残留底物中保留了6.5%)，这表明磷酸盐是从PEP中释放出来的，而不是UMP。从31P和1H{31P}核磁共振谱中获得了确凿的证据，表明核苷酸产物的5‘-位上有一个单独的磷。因此，可以得出结论，5‘-位不是烯醇式丙酮酸的结合部位。为了确定烯醇式丙酮酸部分的实际受体位置，对反应产物进行了分离和纯化，并进行了1H，13C和2D13C，1H-异核核磁共振波谱分析。这一分析来自…
Nikkomycins are produced by several species of Streptomyces and exhibit fungicidal, insecticidal, and acaricidal properties due to their strong inhibition of chitin synthase.[1–4] Structurally, they can be classified as peptidyl nucleosides containing two unusual amino acids, that is, hydroxypyridylhomothreonine and aminohexuronic acid with an N-glycosidically linked base (Scheme 1).[5] Although the chemical structure of nikkomycins has been known since the 1970s, information on their biosynthesis is scarce. Following the cloning of the entire set of structural genes involved in nikkomycin biosynthesis,[6] the enzymatic steps leading to the 4-formyl-4-imidazolin-2-one base were investigated in some detail.[7–9] The peptidyl moiety is synthesized by eleven enzymatic reactions, of which only two have been investigated in depth.[10, 11] The aminohexuronic acid is introduced into the nikkomycin skeleton by the transfer of 5-phosphoribosyl-1-pyrophosphate to the nucleobase (uracil or 4-formyl-4-imidazolin-2-one) followed by addition of an enolpyruvyl moiety from phosphoenolpyruvate (PEP), supposedly to the 5’-hydroxyl group of the ribose.[12, 13] This putative intermediate is then further modified by rather speculative reactions to yield the aminohexuronic acid precursor.[13]Based on amino acid sequence similarity, the nikO gene in the nikkomycin operon appears to encode an enolpyruvyl transferase.[13] This family of enzymes comprises two well-characterized enzymes, that is, 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS, EC 2.5. 1.19) and UDP-N-acetylglucosamine enolpyruvyltransferase (MurA, EC 2.5. 1.7), which catalyze the transfer of the intact enolpyruvyl moiety from PEP to the 5-hydroxyl group of shikimate 3-phosphate and the 3’-hydroxyl group of UDP-N-acetylglucosamine, respectively. Therefore, NikO can be expected to catalyze an enolpyruvyl transfer reaction in nikkomycin biosynthesis. In order to substantiate the role of NikO, we have cloned the gene from Streptomyces tendae Tü901 and heterologously expressed the protein in Escherichia coli. The recombinant protein was purified to homogeneity and analyzed for its activity.[14] Contrary to expectation, no enolpyruvyl transferase activity was found with uridine as the substrate.[13] Instead, UMP was found to serve as a substrate for the enzyme. The enzymatic assay was performed by measuring released phosphate by a colorimetric end-point method and a continuous spectrophotometric enzyme-coupled method that exploits the purine nucleoside phosphorylase reaction.[14–16] The phosphate release observed in these assays is accompanied by the generation of a new nucleotide compound from UMP and PEP, as demonstrated by HPLC analysis.[14] Analysis of the reaction products revealed that phosphate was released stoichiometrically with the formation of the new compound during the enzyme reaction; this indicated that one phosphate group still resided in the nucleotide. This result was substantiated by using 32P-labelled UMP in the enzymatic reaction: 90% of the radiolabel is retained (6.5% in residual substrate); this indicated that the phosphate was released from PEP rather than UMP. Corroborating evidence was obtained from 31P and 1H {31P} NMR spectroscopy, which revealed a single phosphorus attached to the 5’-position in the nucleotide product. Consequently, it can be concluded that the 5’-position is not the site of enolpyruvyl attachment. In order to determine the actual acceptor site of the enolpyruvyl moiety, the reaction product was isolated, purified by RP-HPLC, and subjected to 1H, 13C, and 2D 13C, 1H-heteronuclear NMR spectroscopy. This analysis …