保守残基变异对UGT2B15酶活性影响的结构基础

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Lin Zhang , Xuerong Zhang , Yibing Yang , Jiangyong Gu , Zhongqiu Liu , Caiyan Wang
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引用次数: 0

摘要

UDP葡糖醛酸基转移酶2B15(UGT2B15)是一种重要的II期药物代谢酶,可对包括临床药物和激素在内的各种化合物进行葡糖醛酸化。突变体可能影响葡萄糖醛酸化,导致体内药物代谢中断,降低治疗效果。在这里,我们主要使用葡萄糖醛酸化分析、分子动力学(MD)模拟和X射线衍射方法分析了两个具有代表性的突变体H401P和L446S对UGT2B15活性的影响。L446S的酶活性比野生型明显增加了6倍,但P191L、T374A和H401P的酶活性明显丧失。此外,我们使用MD模拟计算了H401P和L446S在催化过程中的能量变化,结果表明,H401P突变体与氧西泮和UDPGA的自由结合能分别为−30.98±1.00 kcal/mol和−36.42±1.04 kcal/mol,与野生型相比明显增加,表明401位上的突变对催化作用具有关键作用。此外,通过蛋白质晶体学和X射线衍射技术确定了UGT2B15 C-末端结构域L446S的三维结构,结果表明,与野生型相比,S446晶体结构中S446和K410之间形成了一个更多的氢键。等温滴定量热法进一步揭示了携带L446S的UGT2B15的C末端结构域对辅因子UDPGA的Kd值与野生型的值相似。结果表明,H401P和L446S对酶活性的影响机制不同。我们的工作为UGTs催化过程中的方差解释提供了一个有用的机制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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

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

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.

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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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