...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Protein Residues That Control the Reaction Trajectory in S-Adenosylmethionine Radical Enzymes: Mutagenesis of Asparagine 153 and Aspartate 155 in Escherichia coli Biotin Synthase
【24h】

Protein Residues That Control the Reaction Trajectory in S-Adenosylmethionine Radical Enzymes: Mutagenesis of Asparagine 153 and Aspartate 155 in Escherichia coli Biotin Synthase

机译：控制S-腺苷甲硫氨酸自由基酶反应轨迹的蛋白质残基：大肠杆菌生物素合酶中天冬酰胺153和天冬氨酸155的诱变

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Biotin synthase catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) tongenerate the biotin thiophane ring. This reaction is initiated by the reductive cleavage of the sulfoniumncenter of S-adenosyl-L-methionine (AdoMet), generating methionine and a transient 5′-deoxyadenosylnradical that functions as an oxidant by abstracting hydrogen atoms from DTB. Biotin synthase containsna highly conserved sequence motif, YNHNLD, in which Asn153 and Asp155 form hydrogen bonds withnthe ribose hydroxyl groups of AdoMet. In the present work, we constructed four individual site-directednmutations to change each of these two residues in order to probe their role in the active site. We usednmolecular weight filtration assays to show that for most of the mutant enzymes binding of the substratesnwas only slightly affected. In Vitro assays demonstrate that several of the mutant enzymes were able tonreductively cleave AdoMet, but none were able to produce a significant amount of biotin. Several of thenmutants, especially Asn153Ser, were able to produce high levels of the stable intermediate 9-mercaptodethiobiotin.nSome of the mutants, such as Asp155Asn and Asn153Ala, produced instead an alternatenproduct tentatively identified by mass spectrometry as 5′-mercapto-5′-deoxyadenosine, generated by directnattack of the 5′-deoxyadenosyl radical on the [4Fe-4S]2+ cluster. Collectively, these results suggest thatnthe protein residues that form hydrogen bonds to AdoMet and DTB are important for retaining intermediatesnduring the catalytic cycle and for targeting the reactivity of the 5′-deoxyadenosyl radical.
机译：生物素合酶催化硫原子向脱硫生物素（DTB）的氧化加成，从而生成生物素硫烷环。该反应通过还原性裂解S-腺苷-L-甲硫氨酸（AdoMet）的initiated中心而产生，产生甲硫氨酸和一个短暂的5'-脱氧腺苷自由基，该自由基通过从DTB提取氢原子而起氧化剂的作用。生物素合成酶包含一个高度保守的序列基序YNHNLD，其中Asn153和Asp155与AdoMet的核糖羟基形成氢键。在目前的工作中，我们构建了四个单独的定点突变来改变这两个残基中的每一个，以探究它们在活性位点中的作用。我们使用分子量过滤测定法显示，对于大多数突变酶而言，底物的结合仅受到轻微影响。体外测定表明，几种突变酶能够吨还原地切割AdoMet，但没有一种能够产生大量的生物素。当时的几个突变体，尤其是Asn153Ser，能够产生高水平的稳定中间体9-巯基脱硫生物素.n一些突变体，例如Asp155Asn和Asn153Ala，通过质谱法初步鉴定为5'-巯基5'-通过直接攻击[4Fe-4S] 2+簇上的5'-脱氧腺苷基产生的脱氧腺苷。总的来说，这些结果表明，形成与AdoMet和DTB的氢键的蛋白质残基对于保持中间体介导催化循环和靶向5'-脱氧腺苷基的反应性是重要的。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号