CHARACTERIZATION OF HUMAN OHCU DECARBOXYLASE: FROM PROTEIN EXPRESSION TO CATALYTIC ACTIVITY IN URIC ACID METABOLISM

2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase is an
enzyme responsible for the conversion of OHCU to S-allantoin during purine
degradation, a process that is critical for the maintenance of life. However, over the
course of evolution, the selective pressure to maintain this metabolic pathway has
decreased, leading to the loss of enzyme expression in certain organisms such as
hominids, birds and reptiles. As a result, uric acid, which is less soluble than
S-allantoin, accumulates in these organisms as an end product of purine degradation
and can cause hyperuricemia. In humans, hyperuricemia is associated with several
diseases, including diabetes, cardiovascular disease, chronic kidney disease and
gout. Our research group has recently identified gene regions in the human genome
that encode all enzymes required for the conversion of uric acid to S-allantoin,
including urate oxidase (uricase), 5-hydroxyisourate hydrolase (HIUase) and OHCU
decarboxylase. However, protein-coding transcripts were only detected for the
decarboxylase, while the other genes produced unproductive transcripts.
Biochemical and biophysical studies of OHCU decarboxylase have provided
valuable insights for the development of biomolecules to control hyperuricemia. In
this study, we characterized the activity of this metabolic enzyme, which appears to
be impaired by the accumulation of mutations. To increase the stability of the
enzyme, we optimized the expression of the recombinant enzyme in M9 medium.
Hydrolytic sites were identified by MALDI-TOF/MS-MS, which led to the recognition
of key sites for the optimization of the protein design. To evaluate the catalytic
activity of OHCU decarboxylase, we performed UV/VIS and 1D-STD NMR
spectroscopy experiments using uricase from Candida sp. and HIUase from
Herbaspirillum seropedicae in combination with OHCU decarboxylase from Homo
sapiens to produce S-allantoin. Using 1D-STD NMR spectra, we identified the
protons directly involved in the interaction of the enzyme and were able to map the
active site of the enzyme. We traced the formation of each intermediate to the final
product and showed that despite its instability and accumulation of mutations, human
OHCU decarboxylase is functional and has the potential for a biopharmaceutical
application.
This research was supported by grants from Conselho Nacional de Desenvolvimento
Científico e Tecnologico (CNPq/MCTI/FNDCT No 18/2021 grant #409064/2021-1 and
CNPq No September 2022 grant #311420/2022-2) and Fundação de Amparo à
Pesquisa do Estado de Minas Gerais (FAPEMIG; RED-00067-23 and grant

#APQ-00341-22). ACSB was supported by a PhD fellowship from the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; grant
#88882.349264/2019e01; Financial code #001)