Kim Jieun, Kim Yeo-Jin, Choi So Young, Lee Sang Yup, Kim Kyung-Jin
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea.
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Biotechnol J. 2017 Jan;12(1). doi: 10.1002/biot.201600648. Epub 2016 Nov 30.
Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by numerous microorganisms as energy and reducing power storage materials, and have attracted much attention as substitutes for petroleum-based plastics. Here, we report the first crystal structure of Ralstonia eutropha PHA synthase at 1.8 Å resolution and structure-based mechanisms for PHA polymerization. RePhaC1 contains two distinct domains, the N-terminal (RePhaC1 ) and C-terminal domains (RePhaC1 ), and exists as a dimer. RePhaC1 catalyzes polymerization via non-processive ping-pong mechanism using a Cys-His-Asp catalytic triad. Molecular docking simulation of 3-hydroxybutyryl-CoA to the active site of RePhaC1 reveals residues involved in the formation of 3-hydroxybutyryl-CoA binding pocket and substrate binding tunnel. Comparative analysis with other polymerases elucidates how different classes of PHA synthases show different substrate specificities. Furthermore, we attempted structure-based protein engineering and developed a RePhaC1 mutant with enhanced PHA synthase activity.
聚羟基脂肪酸酯（PHA）是由众多微生物合成的天然聚酯，作为能量和还原力储存材料，作为石油基塑料的替代品已引起广泛关注。在此，我们报道了真养产碱杆菌PHA合酶在1.8 Å分辨率下的首个晶体结构以及基于结构的PHA聚合机制。RePhaC1包含两个不同的结构域，即N端（RePhaC1 ）和C端结构域（RePhaC1 ），并以二聚体形式存在。RePhaC1通过使用半胱氨酸-组氨酸-天冬氨酸催化三联体的非连续乒乓机制催化聚合反应。3-羟基丁酰辅酶A与RePhaC1活性位点的分子对接模拟揭示了参与3-羟基丁酰辅酶A结合口袋和底物结合通道形成的残基。与其他聚合酶的比较分析阐明了不同类别的PHA合酶如何表现出不同的底物特异性。此外，我们尝试基于结构的蛋白质工程，并开发了一种具有增强PHA合酶活性的RePhaC1突变体。
