caa a22 4500 605498768 CHVBK 20210128100548.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6520-6 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6520-6 Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions [Elektronische Daten] [Juan Feng, Lu Wang, Hongbo Liu, Xiaolan Yang, Lin Liu, Yanling Xie, Miaomiao Liu, Yunsheng Zhao, Xiang Li, Deqiang Wang, Chang-Guo Zhan, Fei Liao] Bacillus fastidious uricase (BF uricase) containing 322 amino acid residues exhibited high stability under physiological conditions. Its crystal structure was solved to 1.4-Å resolution, showing homotetramer containing two homodimers. After the intersubunit antiparallel β-sheet in its homodimer, each subunit had a total of 18 C-terminus residues forming an α-helix (Q305-A313) and random coil (S314-L322) on surface to bury other two α-helices (I227-T238 and I244-R258). In comparison, reported crystal structures of Arthrobacter globiformis and Aspergillus flavus uricases had atomic coordinates of only some C-terminus residues, while the crystal structures of all the other uricases accessible before September 2014 missed atomic coordinates of all their C-terminus residues, after the intersubunit antiparallel β-sheets. In each homodimer of BF uricase, H-bonds were found between E311 and Y249 and between Y319 and D257; electrostatic interaction networks were found to surround D307 plus R310 and intersubunit R3, K312 plus D257, E318 plus K242, and L322 plus R258. Amino acid mutations that disrupted those interactions when R3 and D307 were reserved caused moderate decreases of activity at pH 9.2 while negligible decreases of activity at pH 7.4, but destroyed stability at pH 7.4 while slightly decreased stability at pH 9.2. Such structural information guided the fusion of 6His-tag to the C-terminus of the mutant L322D with SNSNSN as a linker to reserve the activity and stability. Hence, the folding of the C-terminus residues is crucial for thermal stability of BF uricase under physiological conditions; these new structural insights are valuable for molecular engineering of uricases. Springer-Verlag Berlin Heidelberg, 2015 Bacillus fastidious uricase nationallicence C-terminus residues nationallicence Thermal stability nationallicence Electrostatic interaction center nationallicence Hydrophobic interaction nationallicence Feng Juan Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Wang Lu Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Liu Hongbo Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Yang Xiaolan Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Liu Lin Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, 100093, Beijing, China aut Xie Yanling Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Liu Miaomiao Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Zhao Yunsheng Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Li Xiang Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Wang Deqiang Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Zhan Chang-Guo Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, 40536, Lexington, KY, USA aut Liao Fei Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 7973-7986 0175-7598 99:19<7973 2015 99 253 https://doi.org/10.1007/s00253-015-6520-6 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6520-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Feng Juan Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Wang Lu Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Liu Hongbo Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Yang Xiaolan Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Liu Lin Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, 100093, Beijing, China aut NATIONALLICENCE 700 1- Xie Yanling Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Liu Miaomiao Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Zhao Yunsheng Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Li Xiang Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Wang Deqiang Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 700 1- Zhan Chang-Guo Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, 40536, Lexington, KY, USA aut NATIONALLICENCE 700 1- Liao Fei Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, 400016, Chongqing, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 7973-7986 0175-7598 99:19<7973 2015 99 253