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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structural and mechanistic insight into covalent substrate binding by Escherichia coli dihydroxyacetone kinase
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Structural and mechanistic insight into covalent substrate binding by Escherichia coli dihydroxyacetone kinase

机译：大肠杆菌二羟基丙酮激酶对共价底物结合的结构和机理研究

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摘要

The Escherichia coli dihydroxyacetone (Dha) kinase is an unusual kinase because (ⅰ) it uses the phosphoenolpyruvate carbohydrate: phosphotransferase system (PTS) as the source of high-energy phosphate, (ⅱ) the active site is formed by two subunits, and (ⅲ) the substrate is covalently bound to His218~(K*) of the DhaK subunit. The PTS transfers phosphate to DhaM, which in turn phosphory-lates the permanently bound ADP coenzyme of DhaL. This phos-phoryl group is subsequently transferred to the Dha substrate bound to DhaK. Here we report the crystal structure of the E. coli Dha kinase complex, DhaK-DhaL. The structure of the complex reveals that DhaK undergoes significant conformational changes to accommodate binding of DhaL. Combined mutagenesis and enzymatic activity studies of kinase mutants allow us to propose a catalytic mechanism for covalent Dha binding, phosphorylation, and release of the Dha-phosphate product. Our results show that His56~K is involved in formation of the covalent hemiaminal bond with Dha. The structure of H56N~K with noncovalently bound substrate reveals a somewhat different positioning of Dha in the binding pocket as compared to covalently bound Dha, showing that the covalent attachment to His218~K orients the substrate optimally for phosphoryl transfer. Asp109~K is critical for activity, likely acting as a general base activating the γ-OH of Dha. Our results provide a comprehensive picture of the roles of the highly conserved active site residues of dihydroxyacetone kinases.
机译：大肠杆菌二羟基丙酮（Dha）激酶是一种不常见的激酶，因为（ⅰ）它使用磷酸烯醇丙酮酸碳水化合物：磷酸转移酶系统（PTS）作为高能磷酸盐的来源，（ⅱ）活性位点由两个亚基形成，并且（ ⅲ）底物与DhaK亚基的His218〜（K *）共价结合。 PTS将磷酸盐转移至DhaM，后者再磷酸化DhaL的永久结合ADP辅酶。该磷酰基随后被转移至与DhaK结合的Dha底物。在这里，我们报告了大肠杆菌Dha激酶复合物DhaK-DhaL的晶体结构。该复合物的结构表明，DhaK经历了显着的构象变化，以适应DhaL的结合。激酶突变体的联合诱变和酶促活性研究使我们能够提出共价Dha结合，磷酸化和释放Dha-磷酸盐产物的催化机制。我们的结果表明，His56〜K参与了与Dha的共价半价键的形成。与共价结合的Dha相比，具有非共价结合的底物的H56N〜K的结构揭示了Dha在结合口袋中的位置略有不同，这表明与His218〜K的共价连接使底物最适合进行磷酰基转移。 Asp109〜K对活性至关重要，可能作为激活Dhaγ-OH的一般碱。我们的结果提供了对二羟基丙酮激酶高度保守的活性位点残基的作用的全面描述。

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    Rong Shi; Laura McDonald; Qizhi Cui; Allan Matte; Miroslaw Cygler; Irena Ekiel;

  • 作者单位

    Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6;

    Department of Chemistry and Biochemistry, Concordia University, Montreal, QC, Canada H4B 1R6;

    Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P 2R2;

    Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P 2R2;

    Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6,Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P 2R2;

    Department of Chemistry and Biochemistry, Concordia University, Montreal, QC, Canada H4B 1R6,Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P 2R2;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    DhaK-DhaL complex; kinase mechanism;

    机译：DhaK-DhaL复合体;激酶机制;

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