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首页> 外文学位 >Structural and functional studies of two bacterial PLP-dependent sugar-modifying enzymes.
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Structural and functional studies of two bacterial PLP-dependent sugar-modifying enzymes.

机译：两种细菌PLP依赖性糖修饰酶的结构和功能研究。

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摘要

GDP-colitose and GDP-perosamine are deoxysugars and are constituents of the O-antigens found within various Gram negative bacterial species including Escherichia coli, Vibrio cholerae, and Salmonella enterica. Both sugars are derived from GDP-mannose, which first undergoes an oxidation/reduction reaction by GDP-mannose-4,6-dehydratase to produce GDP-4-keto-6-deoxymannose. ColD removes the 3'-OH group from the hexose moiety of this intermediate, after which additional reactions produce GDP-colitose. GDP-perosamine synthase is an aminotransferase that also acts upon GDP-4-keto-6-deoxymannose, but instead converts the 4'-keto group into an amino group, thereby producing GDP-perosamine. ColD and GDP-perosamine synthase are both pyridoxal-5'-phosphate (PLP)-dependent enzymes, utilize the same substrates, and share an amino acid sequence identity of 23%. Presented in this dissertation is a combined structural and functional study of these two enzymes.;Wild-type structures of both ColD from E. coli O55 and GDP-perosamine synthase from Caulobacter crescentus CB 15 demonstrate that these enzymes belong to the well-characterized aspartate aminotransferase family of PLP-dependent enzymes. Crystal packing interactions and subsequent gel filtration experiments suggest that both enzymes are dimers in their active forms. Examination of the active sites suggests that His 188 and Lys 186 act as the sole general acid/base in ColD and GDP-perosamine synthase, respectively. Site-directed mutant enzyme structures with trapped sugar compounds demonstrate the manner in which these enzymes accommodate their sugar substrates in the active site. On the basis of these structures, a site-directed mutant ColD enzyme was constructed that is no longer a 3-dehydratase, but is instead a 4-aminotransferase. Additionally, by utilizing the wild-type ColD product (GDP-4-keto-3,6-dideoxymannose) as an alternative substrate for GDP-perosamine synthase and subsequently GDP-perosamine acetyltransferase, two novel nucleotide-linked sugar compounds were produced. The ability to alter the function of an enzyme through site-directed mutagenesis and to produce new sugar compounds, as demonstrated in this dissertation, is useful in generating novel sugar-containing therapeutic natural products from existing scaffolds.
机译：GDP-colitose和GDP-perosamine是脱氧糖，是各种革兰氏阴性细菌物种（包括大肠杆菌，霍乱弧菌和肠炎沙门氏菌）中O抗原的组成部分。两种糖均来自GDP-甘露糖，后者首先通过GDP-甘露糖-4,6-脱水酶进行氧化/还原反应，以生产GDP-4-酮-6-脱氧甘露糖。 ColD从该中间体的己糖部分去除了3'-OH基团，此后，其他反应又产生了GDP-colitose。 GDP-过胺合成酶是一种氨基转移酶，其也作用于GDP-4-酮-6-脱氧甘露糖，但是将4'-酮基转化为氨基，从而产生GDP-过胺。 ColD和GDP-过胺合成酶都是吡pyr醛5'-磷酸（PLP）依赖性酶，利用相同的底物，并具有23％的氨基酸序列同一性。本论文提出了这两种酶的结构和功能的组合研究。大肠杆菌O55的ColD和新月形杆菌CB 15的GDP-过氧化胺合酶的野生型结构表明这些酶属于特征明确的天冬氨酸PLP依赖性酶的转氨酶家族。晶体堆积相互作用和随后的凝胶过滤实验表明，两种酶均为活性形式的二聚体。对活性位点的检查表明，His 188和Lys 186分别充当ColD和GDP-过胺合成酶中唯一的通用酸/碱。具有捕获的糖化合物的定点突变酶结构证明了这些酶在活性位点中容纳其糖底物的方式。基于这些结构，构建了定点突变ColD酶，其不再是3-脱水酶，而是4-氨基转移酶。此外，通过利用野生型ColD产物（GDP-4-酮-3,6-二脱氧甘露糖）作为GDP-过胺合成酶和随后的GDP-过胺乙酰转移酶的替代底物，生产了两种新型的核苷酸连接糖化合物。通过定点诱变改变酶的功能并产生新的糖化合物的能力，如本论文所证明的，可用于从现有支架中产生新型的含糖治疗性天然产物。

著录项

  • 作者

    Cook, Paul D.;

  • 作者单位

    The University of Wisconsin - Madison.;

  • 授予单位 The University of Wisconsin - Madison.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 222 p.
  • 总页数 222
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

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