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Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions

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Abstract

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.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (no.30672139), Natural Sciences Foundation of CQ (nos. CSTC2011BA5039 and CSTC2012JJA0057), the Education Ministry of China (grant no.20125503110007), and the National Science Foundation of USA (grant CHE-1111761). We thank scientists in Shanghai Synchrotron Radiation Facility for technical support during collection of data.

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    1. Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China

      Juan Feng, Lu Wang, Hongbo Liu, Xiaolan Yang, Yanling Xie, Miaomiao Liu, Yunsheng Zhao, Xiang Li, Deqiang Wang & Fei Liao

    2. Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China

      Juan Feng & Lin Liu

    3. Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA

      Chang-Guo Zhan

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    Juan Feng, Lu Wang and Hongbo Liu contributed equally to this work.

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    Feng, J., Wang, L., Liu, H. et al. Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions. Appl Microbiol Biotechnol 99, 7973–7986 (2015). https://doi.org/10.1007/s00253-015-6520-6

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