Abstract
Hyperuricemia and gout are increasingly prevalent among global health concerns, necessitating the development of more effective and safer urate level-lowering therapies. As uricase-based therapeutics represent a critical approach for managing refractory gout, challenges related to their activity, stability and immunogenicity need to be addressed. However, the structure-function relationships of uricase remain ambiguous. In this study, we performed structure-guided engineering on Arthrobacter globiformis Uricase (AgUricase), a typical bacterial enzyme characterized by its notable activity and thermostability, to elucidate the structural determinants that govern these critical properties. Through rational structure-based sequence design, we targeted the binding pocket of uric acid, the regions exhibiting structural divergence from the corresponding mammalian uricase, and the predicted channel for substrate entry. We generated fourteen recombinant AgUricase mutants via rational site-directed mutagenesis. Through further tests, we found that a series of AgUricase mutations T67A, K157A, E162G, F163A, L182F, L220P, L222V and F253A obviously reduced the enzymatic activity and/or thermostability. To probe the structural mechanism underlying these functional alterations, we performed molecular dynamics (MD) simulations on both the mutants and the WT enzyme. Notably, the L254N and P259K AgUricase mutants exhibited higher catalytic activity than the WT, although a minor decrease in thermostability was observed. These results demonstrate the crucial role of residues near the substrate access channel in modulating enzymatic function. This study provides new insights into uricase structure-function relationships, which could be fundamentally used for the design of improved uricase-based therapeutics for patients with hyperuricemia and gout.