Biophysical Studies and Challenges in Uric Acid Catabolism Pathway Enzymes

Uric acid is the end product of purine catabolism in humans. In contrast to other organisms, in which uric acid is converted by additional enzymatic steps into more soluble metabolites such as (S)-allantoin, urea, or ammonia, this pathway is absent in humans. Due to its low solubility, an accumulation of uric acid can lead to hyperuricemia, resulting in gout and kidney dysfunction, and it is also considered a risk factor for diabetes, hypertension, and cancer. For decades, the conversion of uric acid to allantoin was attributed exclusively to uricase (Uox), which oxidizes uric acid to 5-hydroxyisourate (HIU). HIU spontaneously decomposes into a racemic mixture of allantoin. However, later studies revealed that two additional enzymes, a hydrolase (Urah) and a decarboxylase (Urad), are involved, which confer stereospecificity to the reaction. The physiological relevance of this stereospecificity is highlighted by studies showing that mice lacking Urah exhibit renal dysfunction, underscoring the essential role of the (S)-allantoin isomer. Despite these findings, few studies have explored the mechanistic details of this pathway. Our aim is to establish a reliable system for the recombinant production and stabilization of Uox, Urah, and Urad, as a prerequisite for mechanistic and structural studies. All proteins were engineered with histidine tags and purified by nickel-affinity chromatography, and the purified proteins displayed enzymatic activity. Our results showed that optimized storage strategies can significantly enhance protein conservation and stability. We also characterized substrates and products of the pathway—uric acid and allantoin—by UV–Vis spectrophotometry (200–350 nm) and high-field nuclear magnetic resonance (600 MHz), providing a foundation for mechanistic analysis.

This work was supported by Conselho Nacional de Desenvolvimento Científico e  Tecnológico (CNPq), by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).