CROSS-SPECIES BIOPHYSICAL ANALYSIS OF 5-HYDROXYISOURATE HYDROLASE: STRUCTURAL, FUNCTIONAL, AND STABILITY INSIGHTS

The enzyme 5-hydroxyisourate hydrolase (HIUase) participates in the uric acid degradation pathway, a process involved in purine catabolism. This enzyme is an homotetrameric protein that is absent in primates. HIUase and human transthyretin share a common evolutionary origin from an ancient gene duplication event, with both adopting a homotetrameric transthyretin-like β-sandwich fold. Leveraging this structural and evolutionary relationship, a new HIUase variant from Herbaspirillum seropedicae was generated through evolution-based protein engineering, showing biotechnological potential for treating hyperuricemic conditions such as gout. The aim of this work is to perform a biophysical characterization of HIUase from Herbaspirillum seropedicae, its new modified form, and the HIUase from Danio rerio, a widely used vertebrate model organism. This comparison seeks to identify structural and functional differences among these proteins and to understand their stability under various physical and chemical conditions. First, we will express and purify the enzymes in a heterologous system. The purified enzymes will be characterized by MALDI-ToF/MS, circular dichroism, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. We will evaluate the biological activity of the enzymes in the uric acid degradation pathway alongside uricase under different pH and temperature conditions to determine the optimal reaction conditions for each enzyme, including kinetic parameters such as K_m, k_cat, and k_cat/K_m. Finally, we will compare the stability profile of each protein under different pH, temperature, and chaotropic agent conditions (e.g., guanidine and urea) using intrinsic fluorescence spectroscopy. Thus, the comparison between the new modified form and the enzymes from other organisms aims to determine whether the engineered HIUase variant retains catalytic activity, using the HIUase from Danio rerio, a vertebrate that naturally expresses a functional form of this enzyme, as a reference for evaluating the modified form's catalytic competence and its potential as a biopharmaceutical agent for the treatment of hyperuricemia.

This work was supported by Conselho Nac. Des. Cient. Tecnologico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).