筛查氧化还原酶辅因子偏好性相关的突变热点
详细信息 查看官网全文
- 英文论文题名：Altering Oxidoreductases in its Cofactor Preference by Hot-site Screening
- 论文作者：刘玉雪 ; 王磊 ; 王雪颖 ; 郭潇佳 ; 赵宗保
- 英文论文作者：Yuxue Liu ; Lei Wang ; Xueying Wang ; Xiaojia Guo ; Zongbao K Zhao ; Dalian institute of chemical physics ; Chinese Academy of Sciences
- 年：2016
- 作者机构：中国科学院大连化学物理研究所生物技术部;
- 论文关键词：生物正交体系 ; 氧化还原酶 ; 亚磷酸脱氢酶 ; 非天然辅酶 ; 突变热点
- 会议召开时间：2016-07-01
- 会议录名称：中国化学会第30届学术年会摘要集-第二十八分会：化学生物学
- 语种：中文
- 分类号：Q55
- 学会代码：ZGHY
- 会议名称：中国化学会第30届学术年会-第二十八分会 ; 化学生物学
- 会议地点：中国辽宁大连
- 主办单位：中国化学会
- 学会名称：中国化学会
- 页数：1
- 文件大小：184k
- 原文格式：D
- 会议级别：全国
摘要
构建非天然辅酶依赖型氧化还原体系,可以为复杂代谢网络提供一种选择性调控氧化还原代谢的新工具。进化筛选对非天然辅酶特异的氧化还原酶,是构建生物正交体系的关键。通常在氧化还原酶的辅酶结合结构域Rossmann fold中选择相关氨基酸残基作为突变位点,所获得的突变型酶在催化活性和辅酶特异性方面尚不能满足需求,亟需建立快速筛查突变热点的方法。本研究结合同源建模、半理性设计和单位点饱和突变技术,设计"热点快速筛查"策略用于高效筛选不同氨基酸位点。利用该策略定向改造亚磷酸脱氢酶(PDH),筛选2790个转化子,完成了对15个位点的筛查,筛选完整度达到99.7%,得到的I151R/P176R和I151R/M207A两种PDH突变体,对非天然辅酶烟酰胺胞嘧啶二核苷酸(NCD)的特异性和催化活性显著提高。通过筛选P176和M207的组合突变文库,获得了对NCD特异性提高5700倍的PDH突变体,为正交体系中NCDH高效再生奠定了基础。本工作对构建其它NCD特异性氧化还原酶具有重要参考价值。
A cofactor-dependent bioorthogonal redox system has excellent characteristics:regulating a particular reaction independent of other metabolic pathways,providing unique tools for synthetic biology and metabolic engineering;and it is crucial to changing cofactor specificity of oxidoreductases.An arginine mutation at the N-terminus of the conserved GXGXXG sequence in Rossmann fold motif can change cofactor specificity to a dedicated abiotic cofactor,nicotinamide cytosine dinucleotide(NCD).But it is not specifically and efficiently for all oxidoreductases.Here,we introduce "hot-site screening" to increase the efficacy of directed evolution.Utilizing single-site saturation mutagenesis to phosphite dehydrogenase(PDH),as a result P176 and M207 were chose as the hot-site.PDH variant with high NCD preference were identified by screening combined saturation libraries.An NCD-specific PDH can be used as cofactors regeneration system improving the bioorthogonal redox system.The strategy was potentially applicable for other NAD-dependent oxidoreductases.
A cofactor-dependent bioorthogonal redox system has excellent characteristics:regulating a particular reaction independent of other metabolic pathways,providing unique tools for synthetic biology and metabolic engineering;and it is crucial to changing cofactor specificity of oxidoreductases.An arginine mutation at the N-terminus of the conserved GXGXXG sequence in Rossmann fold motif can change cofactor specificity to a dedicated abiotic cofactor,nicotinamide cytosine dinucleotide(NCD).But it is not specifically and efficiently for all oxidoreductases.Here,we introduce "hot-site screening" to increase the efficacy of directed evolution.Utilizing single-site saturation mutagenesis to phosphite dehydrogenase(PDH),as a result P176 and M207 were chose as the hot-site.PDH variant with high NCD preference were identified by screening combined saturation libraries.An NCD-specific PDH can be used as cofactors regeneration system improving the bioorthogonal redox system.The strategy was potentially applicable for other NAD-dependent oxidoreductases.
引文
[1]Ji,DB.;Wang,L.;Hou,SH.;et al.J.Am.Chem.Soc.2011,133(51):20857-20862.
[2]王磊,刘武军,刘玉雪,et al.中国科学:生命科学2015,45(10):969-975.
[2]王磊,刘武军,刘玉雪,et al.中国科学:生命科学2015,45(10):969-975.