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首页> 外文学位 >Mechanistic studies of the bifunctional deaminase-reductase RibD and aminoglycoside-N-acetyltransferase from Escherichia coli.
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Mechanistic studies of the bifunctional deaminase-reductase RibD and aminoglycoside-N-acetyltransferase from Escherichia coli.

机译：大肠杆菌双功能脱氨酶还原酶RibD和氨基糖苷-N-乙酰基转移酶的机理研究。

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摘要

Riboflavin is biosynthesized by most microorganisms and plants, while mammals depend entirely on the absorption of this vitamin from the diet to meet their metabolic needs. Therefore, riboflavin biosynthesis is an attractive target for drug design, since appopriate inhibitors would selectively target the microorganism with few side effects in humans. The second and third steps on the pathway are catalyzed by bifunctional proteins containing an N-terminal deaminase and a C-terminal reductase domain, encoded by the E. coli ribD gene. Besides the recent elucidation of the three-dimensional structure of RibD from E. coli and B. subtilis, no detailed enzymatic studies have been performed to date. In the present study we have studied the chemical and kinetic mechanism catalyzed by E. coli RibD. We have demonstrated that the rate of deamination exceeds the rate of reduction and the reductive ring opening occurs by a direct hydride transfer from C4- proR- NADPH to C'-1 of ribose, generating the ribityl moiety of riboflavin.; In the second part of this thesis, we have focused on the studies of aminoglycoside N-acetyltransferases. Clinical resistance to aminoglycosides is generally the result of the expression of enzymes that covalently modify the antibiotic, including N-acetylation. The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from E. coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV, The kinetic mechanism was proposed to be random, based on the observed patterns of dead end inhibition and the non-Michaelis-Menten behavior of certain aminoglycoside substrates. A nanomolar bisubstrate analogue inhibitor was generated enzymatically using AAC(3)-IV, chloroacetyl-CoA and tobramycin. The compound exhibited linear competitive inhibition versus both AcCoA and tobramycin, confirming the rabdom binding of substrates to AAC(3)-IV. The chemical synthesis of other bisubstrate analogs in which the 6'-amino group of aminoglycosides was regioselectively coupled to CoA has been reported. We have tested the first generation of such inhibitors against the Salmonela enterica AAC(6')-Iy. We were able to explain the low affinity constants, as well as the unexpected inhibition patterns, based on the high affinity of the enzyme for its product: the CoA molecule. Indeed, the use of these bisubstrate analogues should provide valuable guidance for the study of other members of the N-acetyltransferase family of enzymes.
机译：核黄素是大多数微生物和植物生物合成的，而哺乳动物则完全依靠从饮食中吸收这种维生素来满足其代谢需要。因此，核黄素的生物合成是药物设计的一个有吸引力的目标，因为适当的抑制剂会选择性地靶向微生物，而对人体的副作用却很少。该途径的第二步和第三步被含有N端脱氨酶和C端还原酶结构域的双功能蛋白催化，该蛋白由大肠杆菌ribD基因编码。除了最近阐明了来自大肠杆菌和枯草芽孢杆菌的RibD的三维结构外，迄今为止尚未进行详细的酶学研究。在本研究中，我们研究了大肠杆菌RibD催化的化学和动力学机理。我们已经证明，脱氨速度超过还原速度，还原环的开环是由氢化物从C4-proR-NADPH直接转移到核糖的C'-1上而产生的，核黄素的核糖基部分。在本文的第二部分，我们着重研究了氨基糖苷N-乙酰基转移酶。临床上对氨基糖苷类药物的耐药性通常是共价修饰抗生素（包括N-乙酰化）的酶表达的结果。来自大肠杆菌的氨基糖苷3-N-乙酰基转移酶AAC（3）-IV表现出非常广泛的氨基糖苷特异性，导致对大量氨基糖苷的耐药性。我们在这里报告的底物特异性的表征和AAC（3）-IV催化的乙酰转移反应的动力学机制，动力学机制被提议为随机的，基于观察到的死角抑制模式和非Michaelis -某些氨基糖苷底物的精神行为。纳摩尔双底物类似物抑制剂使用AAC（3）-IV，氯乙酰辅酶A和妥布霉素酶促生成。该化合物对AcCoA和妥布霉素均表现出线性竞争抑制作用，从而证实了底物与AAC（3）-IV的牢固结合。已经报道了其中氨基糖苷的6'-氨基与CoA区域选择性偶联的其他双底物类似物的化学合成。我们已经测试了第一代针对肠沙门氏菌AAC（6'）-Iy的此类抑制剂。基于酶对其产物CoA分子的高亲和力，我们能够解释其低亲和力常数以及意外的抑制模式。确实，这些双底物类似物的使用应该为研究N-乙酰基转移酶家族的其他成员提供有价值的指导。

著录项

  • 作者

    de Lourdes Borba Magalhaes, Maria.;

  • 作者单位

    Yeshiva University.;

  • 授予单位 Yeshiva University.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 201 p.
  • 总页数 201
  • 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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