Uricolytic Pathway Retention in Great Apes: Recombinant HIUase as a Complementary Enzyme Therapy for Hyperuricemia

Purine degradation in higher primates was evolutionarily interrupted by the inactivation of uricase, a key enzyme of the uricolytic pathway. In humans, this loss leads to uric acid accumulation, causing hyperuricemia and gouty arthritis. Although recombinant uricase is used therapeutically, urate oxidation generates 5-hydroxyisourate (5-HIU), a highly unstable intermediate whose spontaneous, non-enzymatic decay produces a racemic mixture of allantoin, potentially associated with clinical toxicity. The enzyme 5-hydroxyisourate hydrolase (HIUase) specifically converts 5-HIU into OHCU, preventing this uncontrolled decay.While great apes have long been considered to have lost the entire uricolytic pathway downstream of uricase, we found that the HIUase gene is still present in some great ape species, including Pongo abelii, with predicted high sequence and structural similarity to the human ortholog. We hypothesize that recombinant hominoid HIUase could serve as a complementary therapeutic strategy alongside recombinant uricase, increasing metabolic efficiency of urate oxidation and reducing the formation of toxic by-products.In order to study the functionality of this enzyme, we aim to express, purify, and biophysically characterize recombinant HIUase from Pongo abelii (PaHIUase). The gene sequence was commercially synthesized with an N-terminal His-tag and will be recombinantly expressed in Escherichia coli BL21 cells, with optimization of induction parameters to maximize solubility and yield. Purification will be carried out by nickel-affinity chromatography followed by size-exclusion chromatography (SEC) to obtain homogeneous, high-purity samples. Structural stability will be assessed by circular dichroism (CD), and enzymatic activity and kinetic parameters will be monitored by UV-Vis spectrophotometry using 5-HIU generated in situ.We expect to obtain stable, highly pure PaHIUase and to confirm its catalytic activity experimentally. These findings will provide physicochemical insight into the retention and function of uricolytic enzymes in great apes, contributing to the understanding of the structural and catalytic mechanisms underlying this pathway and its potential translational relevance.

This work was supported by the Military Institute of Engineering (IME), by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)