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Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

Ryan, Barry J. and O'Connell, Mary J. and Ó Fágáin, Ciarán (2008) Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase. Biochimie, 90 (9). pp. 1389-1396. ISSN 0300-9084

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The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type's thermal stability (4% and 10% gain in half-life at 50 °C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes.

Item Type:Article (Published)
Uncontrolled Keywords:Ancestral protein; Consensus concept; Protein stabilisation; Recombinant horseradish peroxidase;
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Enzymology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > National Centre for Sensor Research (NCSR)
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Copyright Information:Copyright © 2008 Elsevier Masson SAS
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 License. View License
ID Code:539
Deposited On:02 Sep 2008 17:16 by DORAS Administrator. Last Modified 13 Feb 2017 11:49

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