The structural basis of conserved residue variant effect on enzyme activity of UGT2B15.

ABSTRACT

UDP-glucuronosyltransferase 2B15 (UGT2B15) is a crucial phase II drug-metabolizing enzyme, which glucuronidates various compounds, including clinical drugs and hormones. Mutants might affect glucuronidation, leading to a disruption of drug metabolism in vivo and decrease of therapeutic effect. Here, we mainly analyzed two representative mutants, H401P and L446S, on UGT2B15 activity using glucuronidation assays, molecular dynamic (MD) simulation and X-ray diffraction methods. The enzyme activity of L446S obviously increased six-fold than the wild type, although the enzyme activities of P191L, T374A, and H401P were lost apparently. Furthermore, we used MD simulations to calculate the energy change in the catalytic process of H401P and L446S, and the results indicated the free binding energies of H401P mutant to oxazepam and UDPGA were -30.98 ± 1.00 kcal/mol and -36.42 ± 1.04 kcal/mol, respectively, increased obviously compared to wild type, suggesting the mutation on position 401 had a crucial effect on the catalysis. Moreover, the three-dimensional structure of UGT2B15 C-terminal domain L446S was determined through protein crystallography and X-ray diffraction technology and the results suggested that one more hydrogen bonding between S446 and K410 was formed in the S446 crystal structure, compared to the wild type. Isothermal titration calorimetry assay further revealed the Kd values of C-terminal domain of UGT2B15 harbored L446S towards the cofactor UDPGA was similar to the value of wild type. Above all, our results pointed out that H401P and L446S affected the enzyme activity by different mechanism. Our work provided a helpful mechanism for variance explained in the UGTs catalyzation process.