期刊
ACS CATALYSIS
卷 11, 期 21, 页码 13733-13743出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.1c03950
关键词
tryptophan synthase; distal mutations; allostery; enzyme design; shortest path map; ancestral sequence reconstruction
资金
- Generalitat de Catalunya [2017 SGR-1707]
- Spanish MINECO [PGC2018-102192-B-I00, BES-2015-074964]
- National Research Foundation of Korea (NRF) under the Brain Pool Program [NRF-2021H1D3A2A02038434]
- European Community [H2020-MSCA-IF-2016-753045]
- Juan de la Cierva-Incorporacion fellowship [IJCI-2017-34129]
- European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [ERC-2015-StG-679001]
- Human Frontier Science Program (HFSP) [RGP0054/2020]
- National Research Foundation of Korea [2021H1D3A2A02038434] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
This study focuses on the tryptophan synthase complex and develops a rational approach to identify key amino acid residues for enhancing the activity of TrpB through allosteric effects. Experimental validation shows that the designed TrpB exhibits superior stand-alone activity comparable to enhancements obtained through experimental laboratory evolution. This work indicates that distal active site prediction for enhanced function in computational enzyme design is now achievable.
Allostery is a central mechanism for the regulation of multi-enzyme complexes. The mechanistic basis that drives allosteric regulation is poorly understood but harbors key information for enzyme engineering. In the present study, we focus on the tryptophan synthase complex that is composed of TrpA and TrpB subunits, which allosterically activate each other. Specifically, we develop a rational approach for identifying key amino acid residues of TrpB distal from the active site. Those residues are predicted to be crucial for shifting the inefficient conformational ensemble of the isolated TrpB to a productive ensemble through intra-subunit allosteric effects. The experimental validation of the conformationally driven TrpB design demonstrates its superior stand-alone activity in the absence of TrpA, comparable to those enhancements obtained after multiple rounds of experimental laboratory evolution. Our work evidences that the current challenge of distal active site prediction for enhanced function in computational enzyme design has become within reach.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据