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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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23483 43441 2013 7 PDF Available
Title
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
Abstract

•We detected essential residues for ketoprofen recognition in firefly luciferases.•By replacing these residues was added ketoprofen recognition capacity in HpL.•These residues also affect the enantioselectivity toward ketoprofen.•Characterization of thioesterification properties and MD simulations were performed.•These residues induced the changing of state of hydrogen bonding in the active site.

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 (I350 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.

Graphical abstractWe identified the critical amino acid residues that determine the substrate recognition pattern of thioesterification reaction toward ketoprofen using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) and achieved changing the inactive enzyme to be active and vice versa just by mutating two amino acid residues. These amino acids only affected the enantioselective thioesterification toward ketoprofen and did not influence the bioluminescence colors.Figure optionsDownload full-size imageDownload as PowerPoint slide

Keywords
PmL, firefly luciferase from Pylocoeria miyako; HpL, firefly luciferase from Hotaria parvura; LUC-H, firefly luciferase from Luciola lateralis; PpL, firefly luciferase from Photinus pyralis; LcL, firefly luciferase from Luciola cruciate; LmL, firefly luci
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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
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 168, Issue 3, November 2013, Pages 277–283
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us