Structural Screening And Drug Formation Of Highly Stable Mammalian Uricase

Uricase(EC 1.7.3.3)breaks down uric acid into 5-hydroxyisouric acid and eventually into allantoic acid,which is no longer absorbed by renal tubules and is excreted.Due to a missense mutation of the uricase gene caused by evolution,humans cannot produce active uricase,resulting in hyperuricemia when the concentration of uric acid in the body is too high.Currently,the overall prevalence of hyperuricemia in China is 13.3%,creating a significant market demand.Aspergillus flavus uricase(Rasburicase)has been used to treat hyperuricemia and gout,but its homology with human uricase is low,and it is easy to produce antibodies.Recombinant porcine uricase was developed using Escherichia coli,but it still has high immunogenicity defects.Through multiple sequence alignment of uricase in a variety of mammals,canine uricase with high homology to human uricase was developed,and on this basis,canine uricase mutants with higher homology to humans were constructed.Uricase products with better activity and stability were then selected through various structural screenings.An integrated mammalian uricase,JC,was designed based on the amino acid sequences of more than six mammalian uricases,preserving their highly conserved regions.One or more alternative amino acid compositions were designed in the non-conservative variable region by co-analysis.Three mutants,DC291,DC291-V,and JC291,were designed.By enzymological analysis,the specific activity of JC291 was the highest,which was at least 30% higher than that of wild-type canine uricase.A highly active mutant protein of JC291 was screened.Based on JC291,uricase protein with higher stability was selected by site-specific mutation of amino acid sites.After multiple sequence alignment and feasibility analysis,positions 146,148,245,and 249 of the JC291 amino acid sequence were mutated and named L146 M,S148N,L245 H,and R249 Q,respectively.R249 Q had the highest enzyme activity,which was more than 3% higher than JC291.The activity of L146 M,S148N,and L245 H is not different from JC291,but their stability differs.Through homology modeling and hydrophobic analysis,it was concluded that factors affecting uricase activity are related to the active region of uricase.Mutation sites that affect the active region will increase or decrease enzyme activity.There are many factors affecting the stability of uricase.According to the results of L146 M and S148 N analysis,the hydrophobic action of uricase tetramer both inside and outside will affect the protein structure.R249 Q also indicates that the external α-helical structure had a protective effect on uricase structure.This paper presents a variety of uricase mutants that have been successfully constructed,and rich experimental experience has been accumulated in the gene construction,fermentation expression,and structural properties of uricase.The relationship between activity and stability and the influence of structure on activity and stability have been studied,which provides strong support for future research.