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Design of Bacillus fastidious Uricase Mutants Bearing Long Lagging Phases Before Exponential Decreases of Activities Under Physiological Conditions

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Abstract

Under physiological conditions, Bacillus fastidious uricase (BFU) activity shows negligible lagging phase before the exponential decrease; mutants are thus designed for long lagging phases before exponential activity decreases. On homodimer surface of BFU (4R8X.pdb), the last fragment ANSEYVAL at the C-terminus forms a loop whose Y319 is H-bonded by the buried D257 in the same monomer. Within 1.5 nm from the α-carboxyl group of the last leucine (L322), E30, K26, D257, R258, E311, K312 and E318 from the same monomer plus D126 and K127 from a monomer of the other homodimer generate an electrostatic interaction network. Within 1.5 nm from Y319, D307 and R310 in the same monomer interact with ionized residues around the inter-chain β-sheet in the same homodimer. Mutagenesis of Y319R is designed to strengthen the original interactions and concomitantly generate new electrostatic attractions between homodimers. Under physiological conditions, the mutant V144A/Y319R showed an approximately 4 week lagging phase before the exponential activity decrease, an apparent half-life of activity nearly three folds of mutant V144A, but comparable activity. The introduction of ionizable residues into the C-terminus contacting the other homodimer for additional and/or stronger electrostatic attractions between homodimers may be a universal approach to thermostable mutants of uricases.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (Nos. 81773625 and 30672009). Some mutants reported in this report were included in the invention patent granted in China (Patent No. 201610093491.8, Date of Patent, July 10, 2020). The first three authors contributed equally to this work.

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Author notes

  1. Jiaqi Wang, Luyao Zhang and Jingjing Rao have contributed equally to this work.

Authors and Affiliations

  1. Chongqing Key Laboratory of Medicinal Chemistry & Molecular Pharmacology, Chongqing University of Technology, Chongqing, 400054, China

    Jiaqi Wang, Luyao Zhang, Li Yang & Fei Liao

  2. School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing, 400054, China

    Jiaqi Wang, Luyao Zhang, Li Yang & Fei Liao

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

    Jingjing Rao, Xiaolan Yang & Fei Liao

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  1. Jiaqi Wang
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  2. Luyao Zhang
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  4. Li Yang
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  5. Xiaolan Yang
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Correspondence to Xiaolan Yang or Fei Liao.

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Wang, J., Zhang, L., Rao, J. et al. Design of Bacillus fastidious Uricase Mutants Bearing Long Lagging Phases Before Exponential Decreases of Activities Under Physiological Conditions. Protein J 40, 765–775 (2021). https://doi.org/10.1007/s10930-021-09999-0

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  • DOI: https://doi.org/10.1007/s10930-021-09999-0

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