Glycosylation increases active site rigidity leading to improved enzyme stability and turnover

Krithika Ramakrishnan, Rachel L. Johnson, Samuel D. Winter, Harley L. Worthy, Christopher Thomas, Diana C. Humer, Oliver Spadiut, Sarah H. Hindson, Stephen Wells, Andrew H. Barratt, Georgina E. Menzies, Christopher R. Pudney, D. Dafydd Jones

Research output: Contribution to journalArticlepeer-review

3 Citations (SciVal)

Abstract

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet, the full impact of glycosylation on the protein structure–function relationship, especially in enzymes, is still limited. Here, we show that glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its turnover and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRP's thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its nonglycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data show that glycosylation does not just have a passive effect on HRP stability but can exert long-range effects that mediate the ‘native’ enzyme's activity and stability through changes in inherent dynamics.

Original languageEnglish
Pages (from-to)3812-3827
Number of pages16
JournalFEBS Journal
Volume290
Issue number15
Early online date2 Apr 2023
DOIs
Publication statusPublished - Aug 2023

Bibliographical note

Funding Information:
DDJ would like to thank the BBSRC (grant no. BB/M000249/1) for funding and Wellcome Trust Institutional Strategic Support Fund. RLJ was funded by a Knowledge Economy Skills Scholarship (KESS2 project code 511113). We would like to thank the Protein Technology Hub facility in the School of Biosciences, Cardiff University for access to protein purification and analysis facilities. Molecular dynamic simulations were run on the Hawk facility as part of Supercomputing Wales part‐funded by the European Regional Development Fund (ERDF) via Welsh Government under project code scw1631. CRP would like to thank Agilent Technologies for funding under the ACT‐UR scheme.

Funding Information:
DDJ would like to thank the BBSRC (grant no. BB/M000249/1) for funding and Wellcome Trust Institutional Strategic Support Fund. RLJ was funded by a Knowledge Economy Skills Scholarship (KESS2 project code 511113). We would like to thank the Protein Technology Hub facility in the School of Biosciences, Cardiff University for access to protein purification and analysis facilities. Molecular dynamic simulations were run on the Hawk facility as part of Supercomputing Wales part-funded by the European Regional Development Fund (ERDF) via Welsh Government under project code scw1631. CRP would like to thank Agilent Technologies for funding under the ACT-UR scheme.

Publisher Copyright:
© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Keywords

  • Enzyme enhancment
  • Enzyme rigidity
  • glycosylation
  • molecular dynamics
  • post-translational modification

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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