• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文会议>Conference on biochemical and molecular engineering >THRICE UPON A TIME: THE REPEATED EMERGENCE OF A NOVEL ENZYMATIC FUNCTION FROM AN EVOLVABLE PROTEIN SCAFFOLD
【24h】

THRICE UPON A TIME: THE REPEATED EMERGENCE OF A NOVEL ENZYMATIC FUNCTION FROM AN EVOLVABLE PROTEIN SCAFFOLD

机译：曾经有过一段时光：可进化的蛋白质支架上新酶功能的重复出现

获取原文
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Understanding the emergence of new protein functions from their ancestors is a long-standing challenge in biology and biotechnology; many questions remain unanswered. How can one protein scaffold support multiple distinct functions? How are diverse functions of a superfamily connected? How are major functional switches achieved? Large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies have now begun to provide comprehensive views of functional diversity and evolutionary relationships. Intriguing insights can be gained: promiscuous activities are prevalent and many divergent proteins retain "functional connectivity" via enzyme promiscuity. Interested in the varied biological and biotechnological roles of FMN-dependent "nitroreductase" enzymes (NTRs), we undertook extensive computational and functional analyses to determine sequence, structural and functional relationships2. This large and diverse superfamily contains >80,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions. Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a highly "evolvable" minimal flavin-binding scaffold. To investigate the diverse NTR sequence space for the capacity to catalyze nitroreduction, we synthesized >500 genes and performed high-throughput activity screening to profile 18 in vivo substrates. In vitro kinetic analysis was subsequently performed on 112 enzymes against 32 substrates (vs. 2 nicotinamide cofactors), equating to >7,000 reactions. We demonstrated that only four of the 22 distinct superfamily subgroups display canonical nitroaromatic reductase activities. Eight additional subgroups display occasional promiscuous activities with selected substrates, and 10 subgroups display no nitroreductase activity. Structural analyses revealed the underlying molecular details: nitroreduction has emerged three distinct times in the superfamily via three unique molecular solutions - loop insertions at three different positions in the NTR scaffold, combined with the fixation of key residues, have independently led to functional specialization. These results are now facilitating the rational redesign of the NTR scaffold. Our work provides clues for functional inference for sequences of unknown function, and will aid future efforts to exploit evolvable scaffolds for engineering, and understand the emergence of functional diversity in enzyme superfamilies.
机译：了解其祖先新蛋白质功能的出现是生物学和生物技术领域的一项长期挑战。许多问题仍然没有答案。一种蛋白质支架如何支持多种不同的功能？一个超家族的各种功能如何联系在一起？如何实现主要功能开关？系统地确定酶超家族活性概况的大规模实验方法现在已经开始提供功能多样性和进化关系的全面视图。可以得到有趣的见解：混杂活动普遍存在，许多分歧蛋白通过混杂酶保持“功能连接性”。我们对FMN依赖性“硝基还原酶”酶（NTR）的各种生物学和生物技术作用感兴趣，我们进行了广泛的计算和功能分析，以确定序列，结构和功能之间的关系。这个庞大而多样的超家族包含来自生命所有领域的> 80,000个序列，54个结构和> 10个酶促功能。我们的结果提出了一种进化模型，其中超家族的当代亚组以放射状的方式从高度“可进化的”最小黄素结合支架中分化出来。为了研究多样化的NTR序列空间催化硝基还原的能力，我们合成了> 500个基因并进行了高通量活性筛选，以分析18种体内底物。随后对112种酶针对32种底物（相对于2种烟酰胺辅因子）进行了体外动力学分析，相当于> 7,000次反应。我们证明了22个不同的超家族亚组中只有四个显示出典型的硝基芳香族还原酶活性。另外八个亚组偶尔会与选定的底物发生混杂活动，而另外十个亚组则没有硝基还原酶活性。结构分析揭示了潜在的分子细节：硝基还原通过三种独特的分子溶液在超家族中出现了三个不同的时期-NTR支架中三个不同位置的环插入以及关键残基的固定独立地导致了功能专业化。这些结果现在促进了NTR支架的合理重新设计。我们的工作为未知功能序列的功能推断提供了线索，并将帮助将来开发可进化的支架进行工程研究，并了解酶超家族中功能多样性的出现。

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