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首页> 外文期刊>Journal of Biotechnology >Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues
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Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues

机译：仅仅通过突变两个氨基酸残基即可使用萤火虫（PmL）和小白菜（HpL）萤火虫荧光素酶催化的对映选择性硫酯化反应中的酮洛芬识别的相互转化

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摘要

We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (1350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.
机译：我们确定了使用两种来自萤火虫的萤火虫荧光素酶（PmL）和Hotaria parvura（HpL）的底物识别的关键氨基酸残基，因为这两种萤光素酶对酮洛芬表现出不同的活性。具体而言，PmL可以催化表观的对映选择性硫酯化反应，而HpL则不能。通过比较活性位点周围的氨基酸序列，我们确定了两个酶之间存在差异的两个残基（PmL中的1350和M397和HpL中的F351和S398），替换这些氨基酸导致改变了酮洛芬的识别方式。对于PmL，无活性的HpL被转化为对酮洛芬的活性酶，反之亦然。这些残基也影响对酮洛芬的对映选择性。然而，生物发光颜色不受影响。此外，使用分子动力学计算，这两个氨基酸的取代引起了酮洛芬和S349侧链之间通过活性位点水形成的氢键结合状态的变化。由于S349不被认为会影响色彩调节，因此这些变化会特别引起酶中酮洛芬识别的差异。

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