MECHANISTIC STUDIES OF PROKARYOTIC MANNOSYLTRANSFERASES

关于这种化学品的信息基本上是缺乏的。 糖基转移和掺入的机制 最终靶点(蛋白质、细胞壁)的糖基化模式 和/或抗生素)的发生主要是由于可获得性有限 糖基转移酶及其核苷酸二磷酸糖 底物。虽然这一领域的所有研究基本上都集中在 关于真核转移酶，我们建议原核生物 糖基转移酶是一种丰富的替代来源，也许更好 可访问性。因此，我们建议对两种原核生物进行详细的比较 使用相同底物但不同的甘露糖基转移酶模型 仅在甘露糖转移的立体化学中(字母1产生4或 与Beta1相比，“保留”产生4或“倒置”；Man Talpha4和Man 分别为Tbeta4)。通过多学科方法 (Man Talpha4和Man Tbeta4的高效表达和纯化； 基于人工合成的“非天然”受体的简化检测系统； 化学机理探针，包括底物和溶剂同位素 共价型Man Tα4-和/或的作用、阐明/表征 MAN Tbeta4底物络合物、亲核陷阱和测试 合成底物类似物的催化能力；以及Man Talpha4/Man 包括定点突变在内的Tbeta4结构探针；体外 分子进化；差异标记和/或活性位点定向 亲和力标签)，提议的工作应该有助于定义i)男人 Talpha4和Man Tbeta4化学反应机理和II)人类如何 Talpha4和Man Tbeta4支架控制着这些重要的反应。 以甘露醇转移为模型，所提出的工作将有助于 建立通用糖基转移酶的基础 可以得出机械论的共识。此外，选定的 反应(α/β1产生4甘露醇转移)类似于 在真核细胞N-连接糖蛋白生物合成和 基于这些模型的基于机制的抑制剂应该导致 真核细胞表面糖基化模式改变终止于 N-乙酰-D-氨基葡萄糖(GlcNAc)和Man，将细胞定义为“非我” 从而提供治疗机会。β-甘露糖基连接 也特别难通过化学合成来制备，我们的 拟议的研究可能导致酶替代品的出现。最后， 提出的体外进化/选择方法学应有助于 糖基转移酶的一般合成用途(通过提供 简化的底物和筛选基本上任何 糖基转移事件)，并可能导致未来的细菌细胞 面向设计的治疗性疫苗的表面工程。

There is a fundamental lack of information regarding the chemical mechanisms by which glycosyl transfer and the incorporation of glycosylation patterns into the final target (proteins, cell walls and/or antibiotics) occur due primarily to the restricted availability of glycosyltransferases and their nucleotide diphosphate sugar substrates. While essentially all research in this arena has focused upon eukaryotic transferases, we suggest prokaryotic glycosyltransferases to be a rich alternative source with perhaps better accessibility. Thus we propose a detailed comparison of two prokaryotic model mannosyltransferases which use identical substrates but differ only in the stereochemistry of mannosyltransfer (alpha1 yields 4 or "retaining" versus beta1 yields 4 or "inverting"; Man Talpha4 and Man Tbeta4, respectively). Through a multi-disciplinary approach (overexpression and purification of Man Talpha4 and Man Tbeta4; simplified assay systems based upon synthetic "unnatural" acceptors; chemical mechanistic probes including substrate and solvent isotope effects, elucidation/characterization of covalent Man T alpha4- and/or Man Tbeta4- substrate complexes, nucleophilic traps and testing the catalytic competency of synthetic substrate analogs; and Man Talpha4/Man Tbeta4 structural probes including site-directed mutagenesis; in vitro molecular evolution; differential labeling and/or active site-directed affinity labeling), the proposed work should help define i) the Man Talpha4 and Man Tbeta4 chemical reaction mechanisms and ii) how the Man Talpha4 and Man Tbeta4 scaffold governs these important reactions. Using mannosyl transfer as the model, the proposed work will serve to establish a foundation from which a general glycosyltransferase mechanistic consensus can be derived. In addition, the selected reactions (alpha/beta 1 yields 4 mannosyl transfer) are analogous to those found in eukaryotic N-linked glycoprotein biosynthesis and mechanism-based inhibitors based upon these models should lead to altered eukaryotic cell surface glycosylation patterns terminating in N-acetyl-D-glucosamine (GlcNAc) and Man, defining the cell as "non-self" and thus providing therapeutic opportunities. The beta-mannosyl linkage is also particularly difficult to prepare via chemical synthesis and our proposed studies may lead to enzymatic alternatives. Finally, the presented in vitro evolution/selection methodology should contribute to the general synthetic utility of glycosyltransferases (by providing simplified substrates and the potential to screen for essentially any glycosyl transfer event) and possibly lead to future bacterial cell surface engineering towards designed therapeutic vaccines.