Substrate Specificity of the Macrolide-Glycosylating Enzyme Pair DesVII/DesVIII: Opportunities, Limitations, and Mechanistic Hypotheses
Substrate Specificity of the Macrolide-Glycosylating Enzyme Pair DesVII/DesVIII: Opportunities, Limitations, and Mechanistic Hypotheses
复制标题
DOI：
10.1002/anie.200503195
复制
发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Liu, HW
中科院分区：
文献类型：
--
作者：
Borisova, SA;Zhang, CS;Liu, HW
编码含氨基糖代谢物生物合成的生物合成基因座，它们通常位于氨基糖糖基转移酶基因的直接上游。 [15]因此，DesVIII 最初被认为是这些代谢物中氨基糖部分掺入所必需的。 [6]在本文报道的研究中，我们扩展了已建立的体外 DesVII/DesVIII 测定，以评估该催化对的糖苷配基和糖底物耐受性水平。除了提供一组新的大环内酯外，这项工作还揭示了 DesVII/DesVIII 催化的糖基化对 6-脱氧糖的要求非常严格。更重要的是，这项工作还揭示了 DesVIII 的“增强”不仅限于氨基糖，因此与早期的“氨基糖载体”假说形成鲜明对比。重组 DesVII 蛋白在大肠杆菌中作为 C 端 His6 标记的融合蛋白过量产生，并如前所述进行纯化。 [6] DesVIII 蛋白是在大肠杆菌中产生的，其 N 末端与麦芽糖结合蛋白融合。融合蛋白通过蛋白酶处理进行切割，未标记的 DesVIII 蛋白与 DesVII 一起用于测定。 [16]测试了许多 TDP-糖作为 DesVII/DesVIII 的供体底物，并以天然大环内酯苷元 2（方案 2）作为受体。其中包括葡萄糖 9、半乳糖 10、甘露糖 11 的 TDP 衍生物以及一系列氨基和乙酰氨基己糖 12-18。还检测了在 C3 和 C4、19 和 20 处脱氧的 TDP-糖以及 TDP-木糖 21。由于天然底物去糖胺 (1) 是 6-脱氧己糖，因此还测试了多种 6-脱氧糖 22-27 和 6-脱氧氨基糖 28-31。在所使用的糖核苷酸中，化合物9是市售的，化合物1、[17]、26、[18]和28[17b]是预先化学合成的，化合物30是通过化学酶法制备的，[17b]，化合物23、25、[16]和27[19]是通过酶法合成获得的。其余 16 种 TDP-糖 10-22、24、29、31 是通过工程化的核苷酸转移酶 (Ep) 催化的化学酶途径从各自的糖-1-磷酸原位生成的。 [20]对于每次测定，通过HPLC评估原位TDP-糖形成，随后将反应混合物调节至pH 9，并添加DesVII、DesVIII和2。这
biosynthetic loci encoding for aminosugar-bearing metabolite biosynthesis where they typically reside directly upstream to the aminosugar glycosyltransferase gene.[15] Thus, DesVIII was initially implicated as necessary for the incorporation of aminosugar moieties in these metabolites.[6] In study reported herein, we extend the established in vitro DesVII/DesVIII assay to assess the level of aglycone and sugar substrate tolerance by this catalytic pair. In addition to providing a set of new macrolides, this work reveals a notably stringent 6-deoxysugar requirement for DesVII/DesVIII-catalyzed glycosylation. More importantly, this work also reveals that the DesVIII “boost” is not limited to aminosugars and thus vastly contrasts the earlier “aminosugar carrier” hypothesis.The recombinant DesVII protein was overproduced in E. coli as a C-terminal His6-tagged fusion protein and purified as previously described.[6] The DesVIII protein was produced in E. coli as a fusion with maltose-binding protein at its N terminus. The fusion protein was cleaved by treatment with protease and untagged DesVIII protein was used in the assays together with DesVII.[16] A number of TDP-sugars were tested as donor substrates for DesVII/DesVIII with the native macrolide aglycone 2 (Scheme2) as the acceptor. These include TDP derivatives of glucose 9, galactose 10, mannose 11, and a series of amino and acetamido hexoses 12–18. TDP-sugars deoxygenated at C3 and C4, 19 and 20, as well as TDP-xylose 21 were also examined. As the natural substrate desosamine (1) is a 6-deoxyhexose, a variety of 6-deoxy sugars 22–27 and 6-deoxyaminosugars 28–31 were also tested. Of the sugar nucleotides employed, compound 9 was commercially available, compounds 1,[17] 26,[18] and 28 [17b] were previously synthesized chemically, compound 30 was prepared by a chemoenzymatic method,[17b] and compounds 23, 25,[16] and 27 [19] were obtained through enzymatic synthesis. The remaining 16 TDP-sugars 10–22, 24, 29, 31 were generated in situ through an engineered nucleotidylyltransferase (Ep) catalyzed chemoenzymatic route from the respective sugar-1-phosphates as previously described.[20] For each assay, TDP-sugar formation in situ was assessed by HPLC, the reaction mixture subsequently adjusted to pH 9, and DesVII, DesVIII, and 2 added. The