Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i>
This study details a comprehensive biochemical and structural characterization of exiguolysin, a novel thermolysin-like, caseinolytic peptidase secreted by a marine isolate of <i>Exiguobacterium oxidotolerans</i> strain BW26. Exiguolysin demonstrated optimal proteolytic activity at 37 °C...
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2024-11-01
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| author | Brendan F. Gilmore Tracy A. White Alessandro Busetti Matthew I. McAteer Christine A. Maggs Thomas P. Thompson |
| author_facet | Brendan F. Gilmore Tracy A. White Alessandro Busetti Matthew I. McAteer Christine A. Maggs Thomas P. Thompson |
| author_sort | Brendan F. Gilmore |
| collection | DOAJ |
| description | This study details a comprehensive biochemical and structural characterization of exiguolysin, a novel thermolysin-like, caseinolytic peptidase secreted by a marine isolate of <i>Exiguobacterium oxidotolerans</i> strain BW26. Exiguolysin demonstrated optimal proteolytic activity at 37 °C and pH 3, retaining 85% activity at 50 °C, highlighting its potential stability under broad reaction conditions. SDS-PAGE and LC-MS analysis identified the enzyme as a 32 kDa M4-family metalloprotease. Exiguolysin activity was inhibited by 1,10-phenanthroline, confirming its dependence on metal ions for activity. Zymographic analysis and substrate specificity assays revealed selective hydrolysis of matrix metalloproteinase (MMP) substrates but no activity against elastase substrates. Analysis of the predicted gene sequence and structural predictions using AlphaFold identified the presence and position of HEXXH and Glu-Xaa-Xaa-Xaa-Asp motifs, crucial for zinc binding and catalytic activity, characteristic of ‘Glu-zincins’ and members of the M4 peptidase family. High-throughput screening of a 20 × 20 <i>N</i>-alpha mercaptoamide dipeptide inhibitor library against exiguolysin identified SH-CH<sub>2</sub>-CO-Met-Tyr-NH<sub>2</sub> as the most potent inhibitor, with a <i>K</i>i of 1.95 μM. Notably, exiguolysin selectively inhibited thrombin-induced PAR-1 activation in PC-3 cells, potentially indicating a potential mechanism of virulence in modulating PAR-1 signalling during infection by disarming PARs. This is the first detailed characterization of a peptidase of the M4 (thermolysin) family in the genus <i>Exiguobacterium</i> which may have industrial application potential and relevance as a putative virulence factor. |
| format | Article |
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| issn | 2076-2607 |
| language | English |
| publishDate | 2024-11-01 |
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| series | Microorganisms |
| spelling | doaj-art-7911b752f0fd4664aefe8d512360829b2025-08-20T01:53:54ZengMDPI AGMicroorganisms2076-26072024-11-011211231110.3390/microorganisms12112311Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i>Brendan F. Gilmore0Tracy A. White1Alessandro Busetti2Matthew I. McAteer3Christine A. Maggs4Thomas P. Thompson5Biofilm Research Group, School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UKBiofilm Research Group, School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UKBiofilm Research Group, School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UKBiofilm Research Group, School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UKInstitute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, 19 Chlorine Gardens, Belfast BT9 5DL, UKBiofilm Research Group, School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UKThis study details a comprehensive biochemical and structural characterization of exiguolysin, a novel thermolysin-like, caseinolytic peptidase secreted by a marine isolate of <i>Exiguobacterium oxidotolerans</i> strain BW26. Exiguolysin demonstrated optimal proteolytic activity at 37 °C and pH 3, retaining 85% activity at 50 °C, highlighting its potential stability under broad reaction conditions. SDS-PAGE and LC-MS analysis identified the enzyme as a 32 kDa M4-family metalloprotease. Exiguolysin activity was inhibited by 1,10-phenanthroline, confirming its dependence on metal ions for activity. Zymographic analysis and substrate specificity assays revealed selective hydrolysis of matrix metalloproteinase (MMP) substrates but no activity against elastase substrates. Analysis of the predicted gene sequence and structural predictions using AlphaFold identified the presence and position of HEXXH and Glu-Xaa-Xaa-Xaa-Asp motifs, crucial for zinc binding and catalytic activity, characteristic of ‘Glu-zincins’ and members of the M4 peptidase family. High-throughput screening of a 20 × 20 <i>N</i>-alpha mercaptoamide dipeptide inhibitor library against exiguolysin identified SH-CH<sub>2</sub>-CO-Met-Tyr-NH<sub>2</sub> as the most potent inhibitor, with a <i>K</i>i of 1.95 μM. Notably, exiguolysin selectively inhibited thrombin-induced PAR-1 activation in PC-3 cells, potentially indicating a potential mechanism of virulence in modulating PAR-1 signalling during infection by disarming PARs. This is the first detailed characterization of a peptidase of the M4 (thermolysin) family in the genus <i>Exiguobacterium</i> which may have industrial application potential and relevance as a putative virulence factor.https://www.mdpi.com/2076-2607/12/11/2311<i>Exiguobacterium oxidotolerans</i>metalloproteasethermolysinprotease-activated receptors |
| spellingShingle | Brendan F. Gilmore Tracy A. White Alessandro Busetti Matthew I. McAteer Christine A. Maggs Thomas P. Thompson Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i> Microorganisms <i>Exiguobacterium oxidotolerans</i> metalloprotease thermolysin protease-activated receptors |
| title | Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i> |
| title_full | Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i> |
| title_fullStr | Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i> |
| title_full_unstemmed | Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i> |
| title_short | Exiguolysin, a Novel Thermolysin (M4) Peptidase from <i>Exiguobacterium oxidotolerans</i> |
| title_sort | exiguolysin a novel thermolysin m4 peptidase from i exiguobacterium oxidotolerans i |
| topic | <i>Exiguobacterium oxidotolerans</i> metalloprotease thermolysin protease-activated receptors |
| url | https://www.mdpi.com/2076-2607/12/11/2311 |
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