MECHANISTIC STUDIES OF PROKARYOTIC MANNOSYLTRANSFERASES

原核甘露糖基转移酶的机制研究

• 批准号: 6019500
• 负责人: Jon Scott Thorson
• 金额: $ 19.66万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019500
• 关键词: active sites; affinity labeling; enzyme mechanism; enzyme model; enzyme substrate analog; hexosyltransferase; mannose; microorganism metabolism; molecular cloning; prokaryote; protein purification; site directed mutagenesis; spectrometry; stable isotope; stereochemistry; transfection

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.

关于该化学品的资料基本上缺乏 糖基转移和掺入的机制 糖基化模式进入最终目标（蛋白质、细胞壁 和/或抗生素）主要由于有限的可用性而发生 糖基转移酶及其核苷酸二磷酸糖 印刷受体. 虽然在这个竞技场的所有研究基本上都集中在 在真核转移酶，我们建议原核 糖基转移酶是一种丰富的替代来源， 可访问性。 因此，我们提出了一个详细的比较两个原核生物 使用相同底物但不同的模式甘露糖基转移酶 仅在甘露糖基转移的立体化学中（α 1产生4或 “保留”与β 1产生4或“反转”; Man Talpha 4和Man Tbeta4）。 通过多学科方法 （Man T α 4和Man T β 4的过表达和纯化; 基于合成的“非天然”受体的简化的测定系统; 包括底物和溶剂同位素的化学机理探针 共价Man T α 4-和/或 Man Tbeta 4-底物复合物，亲核陷阱和测试 合成底物类似物的催化能力;和Man Talpha 4/Man Tbeta 4结构探针，包括定点诱变;体外 分子进化;差异标记和/或活性位点定向 亲和标记），拟议的工作应有助于定义i）人 Talpha 4和Man Tbeta 4的化学反应机制和ii）人如何 Talpha 4和Man Tbeta 4支架控制着这些重要的反应。 利用甘露糖基转移作为模型，所提出的工作将有助于 为通用糖基转移酶 可以得出机械共识。此外，被选中的 反应（α/β 1产生4甘露糖基转移）类似于 在真核N-连接糖蛋白生物合成中发现的那些， 基于这些模型的基于机制的抑制剂应该导致 改变的真核细胞表面糖基化模式终止于 N-乙酰-D-葡萄糖胺（GlcNAc）和Man，将细胞定义为“非自我” 从而提供治疗机会。 β-甘露糖键 也特别难以通过化学合成制备， 拟议的研究可能会导致酶的替代品。 最后 提出的体外进化/选择方法应有助于 糖基转移酶的一般合成效用（通过提供 简化的基底和筛选基本上任何 糖基转移事件），并可能导致未来细菌细胞 表面工程设计的治疗性疫苗。