BIOINFORMATIC ANALYSIS AND BIOPHYSICAL CHARACTERIZATION OF HOMO SAPIENS OHCU DECARBOXYLASE: STRUCTURAL, FUNCTIONAL, AND ENZYME ENGINEERING IMPLICATIONS

In hominids, the final product of purine catabolism is uric acid, unlike in other placental mammals where uric acid is degraded into (S)-allantoin through a series of reactions catalyzed by urate oxidase (uricase), HIU hydrolase (HIUase), and OHCU decarboxylase. In H. sapiens, studies on the uric acid degradation pathway revealed that mutations in the uricase gene have led to reduced positive selection pressure for maintaining the gene regions of other enzymes in the pathway. The lack of expression of these enzymes can lead, in some situations, to the accumulation of uric acid. Despite the apparent absence of the proteins in H. sapiens, our analyses showed transcription of the gene regions for uricase (UOX), HIUase (URAHP), OHCU decarboxylase (URAD), and allantoicase (ALLC), with URAD and ALLC producing protein-coding transcripts. Given the potential presence of protein products from URAD and ALLC, we evaluated the possibility of producing active enzymes. Alignments of the primary structure indicated that the protein product of URAD, with 52.02% identity to the Danio rerio OHCU decarboxylase, appeared promising. Furthermore, analysis using PFstats, which assesses positional conservation and residue coevolution networks, showed the conservation of structurally and functionally important residue groups in the URAD protein product. We subsequently modeled the tertiary structures using AlphaFold, Modeller, and i-Tasser tools. The monomeric subunit structures were analyzed and compared to the D. rerio OHCU decarboxylase, yielding RMSD (Root Mean Square Deviation) values of 0.692 Å, 0.221 Å, and 0.301 Å, respectively. We then expressed recombinant H. sapiens OHCU decarboxylase in a heterologous system. Upon obtaining the purified recombinant protein, its sequence was confirmed using mass spectrometry. Enzymatic assays, conducted with the recombinant H. sapiens OHCU decarboxylase, recombinant uricase from Candida sp., and recombinant HIUase from Herbaspirillum seropedicae, confirmed its OHCU decarboxylase activity. Structural characterization using circular dichroism (CD) and nuclear magnetic resonance (NMR) indicated that the produced protein's structure, predominantly composed of α-helices, is consistent with other OHCU decarboxylases reported in the literature. These results provide valuable insights for future protein engineering studies aimed at designing enzymes that display improved catalytic efficiency and stability.