Crystal Structure Determination of Nucleotide-sugar Binding Domain of Human UDP-glucuronosyltransferases 2B10.

BACKGROUND: UDP-glucuronosyltransferases (UGTs) play a crucial role in maintaining endobiotic homeostasis and metabolizing xenobiotic compounds, particularly clinical drugs. However, the detailed catalytic mechanism of UGTs has not been fully elucidated due to the limited availability of reliable protein structures. Determining the catalytic domain of human UGTs has proven to be a significant challenge, primarily due to the difficulty in purifying and crystallizing the full-length protein. OBJECTIVES: This study focused on the human UGT2B10 C-terminal cofactor binding domain, aiming to provide structural insights into the fundamental catalytic mechanisms. METHODS: In this study, the C-terminal sugar-donor binding domain of human UGT2B10 was purified and crystallized using the vapor-diffusion method. The resulting UGT2B10 CTD crystals displayed high-quality diffraction patterns, allowing for data collection at an impressive resolution of 1.53 Å using synchrotron radiation. Subsequently, the structure of the UGT2B10 CTD was determined using the molecule replacement method with a homologous structure. RESULTS: The crystals were monoclinic, belonging to the space C2 with unit-cell parameters a = 85.90 Å, b = 58.39 Å, c = 68.87 Å, α = γ = 90°, and ß = 98.138°. The Matthews coefficient VM was determined to be 2.24 Å3 Da-1 (solvent content 46.43%) with two molecules in the asymmetric unit. CONCLUSION: The crystal structure of UGT2B10 CTD was solved at a high resolution of 1.53 Å, revealing a conserved cofactor binding pocket. This is the first study determining the C-terminal cofactor binding domain of human UGT2B10, which plays a key role in additive drug metabolism.

A potential implication of UDP-glucuronosyltransferase 2B10 in the detoxification of drugs used in pediatric hematopoietic stem cell transplantation setting: an in silico investigation.

BACKGROUND: Sinusoidal occlusion syndrome (SOS) is a potentially severe complication following hematopoietic stem cell transplantation (HSCT) in pediatric patients. Treatment related risk factors such as intensity of conditioning, hepatotoxic co-medication and patient related factors such as genetic variants predispose individuals to develop SOS. The variant allele for SNP rs17146905 in UDP-glucuronosyl transferase 2B10 (UGT2B10) gene was correlated with the occurrence of SOS in an exome-wide association study. UGT2B10 is a phase II drug metabolizing enzyme involved in the N-glucuronidation of tertiary amine containing drugs. METHODS: To shed light on the functionality of UGT2B10 enzyme in the metabolism of drugs used in pediatric HSCT setting, we performed in silico screening against custom based library of putative ligands. First, a list of potential substrates for in silico analysis was prepared using a systematic consensus-based strategy. The list comprised of drugs and their metabolites used in pediatric HSCT setting. The three-dimensional structure of UGT2B10 was not available from the Research Collaboratory Structural Bioinformatics - Protein Data Bank (RCSB - PDB) repository and thus we predicted the first human UGT2B10 3D model by using multiple template homology modeling with MODELLER Version 9.2 and molecular docking calculations with AutoDock Vina Version 1.2 were implemented to quantify the estimated binding affinity between selected putative substrates or ligands and UGT2B10. Finally, we performed molecular dynamics simulations using GROMACS Version 5.1.4 to confirm the potential UGT2B10 ligands prioritized after molecular docking (exhibiting negative free binding energy). RESULTS: Four potential ligands for UGT2B10 namely acetaminophen, lorazepam, mycophenolic acid and voriconazole n-oxide intermediate were identified. Other metabolites of voriconazole satisfied the criteria of being possible ligands of UGT2B10. Except for bilirubin and 4-Hydroxy Voriconazole, all the ligands (particularly voriconazole and hydroxy voriconazole) are oriented in substrate binding site close to the co-factor UDP (mean ± SD; 0.72 ± 0.33 nm). Further in vitro screening of the putative ligands prioritized by in silico pipeline is warranted to understand the nature of the ligands either as inhibitors or substrates of UGT2B10. CONCLUSIONS: These results may indicate the clinical and pharmacological relevance UGT2B10 in pediatric HSCT setting. With this systematic computational methodology, we provide a rational-, time-, and cost-effective way to identify and prioritize the interesting putative substrates or inhibitors of UGT2B10 for further testing in in vitro experiments.