Unlocking the Stereoselectivity and Substrate Acceptance of Enzymes: Proline-Induced Loop Engineering Test
Dr. Ge Qu
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorYuexin Bi
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
University of Science and Technology of China, Hefei, 230027 China
Search for more papers by this authorBeibei Liu
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
Search for more papers by this authorJunkuan Li
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
Department of Chemistry, School of Science, Tianjin University, Tianjin, 300072 China
Search for more papers by this authorXu Han
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorDr. Weidong Liu
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorYingying Jiang
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Search for more papers by this authorZongmin Qin
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Search for more papers by this authorCorresponding Author
Prof. Dr. Zhoutong Sun
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorDr. Ge Qu
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorYuexin Bi
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
University of Science and Technology of China, Hefei, 230027 China
Search for more papers by this authorBeibei Liu
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
Search for more papers by this authorJunkuan Li
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
Department of Chemistry, School of Science, Tianjin University, Tianjin, 300072 China
Search for more papers by this authorXu Han
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorDr. Weidong Liu
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorYingying Jiang
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Search for more papers by this authorZongmin Qin
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
University of Chinese Academy of Sciences, Beijing, 100049 China
Search for more papers by this authorCorresponding Author
Prof. Dr. Zhoutong Sun
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
National Technology Innovation Center of Synthetic Biology, Tianjin, 300308 China
Search for more papers by this authorAbstract
Protein stability and evolvability influence each other. Although protein dynamics play essential roles in various catalytically important properties, their high flexibility and diversity makes it difficult to incorporate such properties into rational engineering. Therefore, how to unlock the potential evolvability in a user-friendly rational design process remains a challenge. In this endeavor, we describe a method for engineering an enantioselective alcohol dehydrogenase. It enables synthetically important substrate acceptance for 4-chlorophenyl pyridine-2-yl ketone, and perfect stereocontrol of both (S)- and (R)-configured products. Thermodynamic analysis unveiled the subtle interaction between enzyme stability and evolvability, while computational studies provided insights into the origin of selectivity and substrate recognition. Preparative-scale synthesis of the (S)-product (73 % yield; >99 % ee) was performed on a gram-scale. This proof-of-principle study demonstrates that interfaced proline residues can be rationally engineered to unlock evolvability and thus provide access to new biocatalysts with highly improved catalytic performance.
Conflict of interest
This work has been included in a patent application by the Tianjin Institute of Industrial Biotechnology.
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