| Autor: |
James E. Pease, Carl Haslam, Robert Edwards, Andrew P. Fosberry, Danuta Mossakowska, Emma J. Jones, Richard A. Lawrenson, Mark Reglinski, Max Pearson, Shiranee Sriskandan |
| Rok vydání: |
2021 |
| Předmět: |
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| DOI: |
10.1101/2021.11.14.468564 |
| Popis: |
The Streptococcus pyogenes cell envelope protease (SpyCEP) is a vital virulence factor in streptococcal pathogenesis. Despite its key role in disease progression and strong association with invasive disease, little is known about the enzymatic function beyond the ELR+ CXC chemokine substrate range. We utilised multiple SpyCEP constructs to interrogate the protein domains and catalytic residues necessary for enzyme function. We leveraged high-throughput mass spectrometry to describe the Michaelis-Menton parameters of active SpyCEP, revealing a Michaelis-Menton constant (KM) of 53.49 nM and a turnover of 1.34 molecules per second, for the natural chemokine substrate CXCL8.Unexpectedly, we found that an N-terminally-truncated SpyCEP C-terminal construct consisting of only the H279 and S617 catalytic dyad had specific CXCL8 cleaving activity, albeit with a reduced substrate turnover of 2.45 molecules per hour, representing a ~2000-fold reduction in activity. In contrast, the KM of the C-terminal SpyCEP construct and full-length enzyme did not differ. We conclude that the SpyCEP C-terminus plays a key role in substrate binding and recognition with key implications for both current and future streptococcal vaccine designs. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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