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

Jiaqi Wang, Luyao Zhang,Jingjing Rao,Li Yang,Xiaolan Yang,Fei Liao

The Protein Journal(2021)

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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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Key words
Bacillus fastidious uricase, Thermostability, Electrostatic interaction network, Ion pair, Lagging phase
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