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Sharp Tools Make Good Work: The Early Success of ASCEM

Dr. Ming-Daw Tsai, Director of Academia Sinica Cryo-EM Center (ASCEM) and Distinguished Research Fellow of Institute of Biological Chemistry, collaborates with Dr. Chin-Yu Chen, an assistant professor from Department of Life Science National Central University, in using the state of the art technology, cryo-electron microscopy (cryo-EM), to uncover the structural bases of pH effect and cofactor bispecificity of ketol-acid reductoisomerase from archaea Sulfolobus solfataricus. The result is published in the Journal of the American Chemical Society on April 17 and featured on the Journalcover.

Cryo-EM, a single particle protein structure technology, was selected as the 2015 most important research method by Nature. Three frontier researchers of cryo-EM were jointly awarded the 2017 Nobel Prize in Chemistry. In response to these developments, Academia Sinica and Taiwan Protein Project (TPP) established ASCEM by purchasing two cutting-edge cryo-EM instruments, Talos Arctica and Titan Krios, in 2017. After setting up, ASCEM started operation in September 2018, with a goal of raising the global competiveness of Taiwan in life science research.

Under the effort of the cryo-EM team especially Dr. Yuan-Chih Chang, an Associate Research Scientist, the collaborative project of Dr. Tsai and Dr. Chen came to fruition quickly in less than a year. This is the first paper in using high-resolution cryo-EM to address specific problems in enzymology. The study determined the structures of ketol-acid reductoisomerase (KARI) at different pH values, and in complex with different nicotinamide cofactors (NADH or NADPH) and a substrate analog. The results led to elucidation of the structural basis of pH effect and cofactor bispecificity of the enzyme.

KARI is the enzyme in the second step of the branched-chain amino acids (BCAAs) biosynthetic pathway. The demand for essential BCAAs, for use in animal feed and human supplements, is currently estimated to exceed 1,500 tons per year, making it a multimillion-dollar business. Recently the BCAA pathway has been engineered in Escherichia coli for isobutanol production as a promising second-generation biofuel by Prof. James C. Liao, President of Academia Sinica, and Prof. Frances H. Arnold, 2018 Nobel Prize Laureate in Chemistry, from California Institute of Technology. In addition, because KARI with preference for NADH has a great advantage in industrial application, Prof. Arnold also endeavors in modifying the enzyme to reverse its cofactor dependence from NADPH to NADH. Here, our cryo-EM structures provide insight into the enzyme’s cofactor bispecificity. The result has a high potential for industrial application, including isobutanol production. The study also demonstrates the powerfulness of cryo-EM in enzymology research.

As Dr. Ming-Daw Tsai says, “The establishment of ASCEM is perhaps three years too late relative to the frontier of the world, but we have done our best to catch up. The operation is already first-rate, and the results are coming out. If biologists can make good use of this facility, the life science research of Academia Sinica can secure the first tier worldwide.” The initial success of ASCEM is also a big encouragement to TPP, which has been supporting cryo-EM studies since the very beginning, along with other areas of protein research with disease relevance, including the development of protein therapeutics.

The publication is entitled Use of Cryo-EM to Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-acid Reductoisomerase.

The Full research article: https://pubs.acs.org/doi/10.1021/jacs.9b01354

Authors : Chen CY, Chang YC, Lin BL, Lin KF, Huang CH, Hsieh DL, Ko TP, Tsai MD

For ASCEM website, please refer to http://cryoem.ibc.sinica.edu.tw

For Taiwan Protein Project (TPP), please refer to http://tpp.ibc.sinica.edu.tw/

Updated : 2019.04.18 (Edited from Academia Sinica press release)

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Image: The result is published in the Journal of the American Chemical Society on April 17 and featured on the Journal cover.

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Image: the difference between high-resolution cryo-EM(left) and low-resolution cryo-EM(right)

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Image: raw images from cryo-electron microscopy

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Academia Sinica and Taiwan Protein Project (TPP) established ASCEM by purchasing two cutting-edge cryo-EM instruments, Talos Arctica (right) and Titan Krios (left), in 2017.