BIOPHYSICAL COMPARISON BETWEEN THE 5-HYDROXY-ISOURATE HYDROLYASE (HIUase) ENZYME FROM Herbaspirillum seropedicae AND A NEW FORM MODIFIED BY EVOLUTION-BASED PROTEIN ENGINEERING

The enzyme 5-hydroxyisourate hydrolase (HIUase) is involved in the breakdown of uric acid, a metabolite produced during purine catabolism. This enzyme is a homotetrameric protein that is not found in hominids. Based on its structural similarity to human transthyretin, a thyroid hormone and retinol carrier in human blood, a new form of HIUase, modified by evolution-based protein engineering was produced from Herbaspirillum seropedicae, showing biotechnological potential for the treatment of hyperuricemic diseases such as chronic refractory gout. Therefore, the aim of this work is the biophysical characterization of HIUase from Herbaspirillum seropedicae, this new modified form and HIUase from Danio rerio, a widely used vertebrate model organism. This comparison aims to identify structural and functional differences between these proteins, understand their stability under different physical and chemical conditions and, thus, obtain important data that can guide the development of a biopharmaceutical formulation and its production process. Initially, we intend to express and purify the enzymes in a heterologous system. Subsequently, the purified enzyme will be characterized by MALDI-ToF/MS, circular dichroism, and sodium dodecyl sulfate acrylamide gel electrophoresis. The biological activity of the enzymes in the uric acid degradation pathway with uric acid oxidase is studied in different conditions by using  different spectroscopic approaches, like intrinsic fluorescence, low angle X ray scattering and differential scanning calorimetry. Altogether, this work will provide key insights into the structural and functional features of HIUases, paving the way for their potential application in novel therapeutic strategies against hyperuricemic diseases. 

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).