Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei
IntroductionS-layer anchoring in Paenibacillus alvei is enabled by a non-covalent interaction between an S-layer homology domain trimer and a secondary cell wall polymer (SCWP), ensuring the structural integrity of the bacterial cell wall. Within the SCWP repeat, pyruvylated ManNAc serves as the lig...
Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2024-09-01
|
| Series: | Frontiers in Molecular Biosciences |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2024.1470989/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850261942534406144 |
|---|---|
| author | Cordula Stefanović Max S. G. Legg Nick Mateyko Jakob J. Ender Tea Kuvek Chris Oostenbrink Christina Schäffer Stephen V. Evans Fiona F. Hager-Mair |
| author_facet | Cordula Stefanović Max S. G. Legg Nick Mateyko Jakob J. Ender Tea Kuvek Chris Oostenbrink Christina Schäffer Stephen V. Evans Fiona F. Hager-Mair |
| author_sort | Cordula Stefanović |
| collection | DOAJ |
| description | IntroductionS-layer anchoring in Paenibacillus alvei is enabled by a non-covalent interaction between an S-layer homology domain trimer and a secondary cell wall polymer (SCWP), ensuring the structural integrity of the bacterial cell wall. Within the SCWP repeat, pyruvylated ManNAc serves as the ligand and the UDP-GlcNAc-2-epimerase MnaA supplies UDP-ManNAc to SCWP biosynthesis.MethodsTo better understand SCWP biosynthesis and identify strategies for inhibiting pathogens with comparable cell wall architecture, like Bacillus anthracis, MnaA and rational variants were produced in E. coli and their kinetic constants determined. The effect of UDP-GlcNAc as a predicted allosteric activator and tunicamycin as a potential inhibitor of MnaA was tested in vitro supported by molecular docking experiments. Additionally, wild-type MnaA was crystallized.ResultsWe present the crystal structure of unliganded P. alvei MnaA resolved at 2.20 Å. It adopts a GT-B fold consistent with other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerases. A comparison of amino acid sequences reveals conservation of putative and known catalytic and allosteric-site residues in MnaA, which was confirmed through analysis of Q42A, Q69A, E135A and H241A MnaA variants. The kinetic parameters KM and kcat of MnaA were determined to be 3.91 mM and 33.44 s-1 for the forward, and 2.41 mM and 6.02 s-1 for the reverse reaction. While allosteric regulation by UDP-GlcNAc has been proposed as a mechanism for enzyme activation, UDP-GlcNAc was not found to be essential for UDP-ManNAc epimerization by P. alvei MnaA. However, the reaction rate doubled upon addition of 5% UDP-GlcNAc. Unexpectedly, the UDP-GlcNAc analog tunicamycin did not inhibit MnaA. Molecular docking experiments comparing tunicamycin binding of P. alvei MnaA and Staphylococcus aureus MnaA, which is inhibited by tunicamycin, revealed different residues exposed to the antibiotic excluding, those at the predicted allosteric site of P. alvei MnaA, corroborating tunicamycin resistance.ConclusionThe unliganded crystal structure of P. alvei MnaA reveals an open conformation characterized by an accessible cleft between the N- and C-terminal domains. Despite the conservation of residues involved in binding the allosteric activator UDP-GlcNAc, the enzyme is not strictly regulated by the substrate. Unlike S. aureus MnaA, the activity of P. alvei MnaA remains unaffected by tunicamycin. |
| format | Article |
| id | doaj-art-4f474be9dae84b649ebadd47ae0ebb1b |
| institution | OA Journals |
| issn | 2296-889X |
| language | English |
| publishDate | 2024-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj-art-4f474be9dae84b649ebadd47ae0ebb1b2025-08-20T01:55:18ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2024-09-011110.3389/fmolb.2024.14709891470989Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alveiCordula Stefanović0Max S. G. Legg1Nick Mateyko2Jakob J. Ender3Tea Kuvek4Chris Oostenbrink5Christina Schäffer6Stephen V. Evans7Fiona F. Hager-Mair8Department of Chemistry, Institute of Biochemistry, NanoGlycobiology Research Group, Universität für Bodenkultur Wien, Vienna, AustriaDepartment of Biochemistry & Microbiology, University of Victoria, Victoria, BC, CanadaDepartment of Biochemistry & Microbiology, University of Victoria, Victoria, BC, CanadaDepartment of Chemistry, Institute of Biochemistry, NanoGlycobiology Research Group, Universität für Bodenkultur Wien, Vienna, AustriaDepartment of Material Sciences and Process Engineering, Institute of Molecular Modeling and Simulation, Universität für Bodenkultur Wien, Vienna, AustriaDepartment of Material Sciences and Process Engineering, Institute of Molecular Modeling and Simulation, Universität für Bodenkultur Wien, Vienna, AustriaDepartment of Chemistry, Institute of Biochemistry, NanoGlycobiology Research Group, Universität für Bodenkultur Wien, Vienna, AustriaDepartment of Biochemistry & Microbiology, University of Victoria, Victoria, BC, CanadaDepartment of Chemistry, Institute of Biochemistry, NanoGlycobiology Research Group, Universität für Bodenkultur Wien, Vienna, AustriaIntroductionS-layer anchoring in Paenibacillus alvei is enabled by a non-covalent interaction between an S-layer homology domain trimer and a secondary cell wall polymer (SCWP), ensuring the structural integrity of the bacterial cell wall. Within the SCWP repeat, pyruvylated ManNAc serves as the ligand and the UDP-GlcNAc-2-epimerase MnaA supplies UDP-ManNAc to SCWP biosynthesis.MethodsTo better understand SCWP biosynthesis and identify strategies for inhibiting pathogens with comparable cell wall architecture, like Bacillus anthracis, MnaA and rational variants were produced in E. coli and their kinetic constants determined. The effect of UDP-GlcNAc as a predicted allosteric activator and tunicamycin as a potential inhibitor of MnaA was tested in vitro supported by molecular docking experiments. Additionally, wild-type MnaA was crystallized.ResultsWe present the crystal structure of unliganded P. alvei MnaA resolved at 2.20 Å. It adopts a GT-B fold consistent with other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerases. A comparison of amino acid sequences reveals conservation of putative and known catalytic and allosteric-site residues in MnaA, which was confirmed through analysis of Q42A, Q69A, E135A and H241A MnaA variants. The kinetic parameters KM and kcat of MnaA were determined to be 3.91 mM and 33.44 s-1 for the forward, and 2.41 mM and 6.02 s-1 for the reverse reaction. While allosteric regulation by UDP-GlcNAc has been proposed as a mechanism for enzyme activation, UDP-GlcNAc was not found to be essential for UDP-ManNAc epimerization by P. alvei MnaA. However, the reaction rate doubled upon addition of 5% UDP-GlcNAc. Unexpectedly, the UDP-GlcNAc analog tunicamycin did not inhibit MnaA. Molecular docking experiments comparing tunicamycin binding of P. alvei MnaA and Staphylococcus aureus MnaA, which is inhibited by tunicamycin, revealed different residues exposed to the antibiotic excluding, those at the predicted allosteric site of P. alvei MnaA, corroborating tunicamycin resistance.ConclusionThe unliganded crystal structure of P. alvei MnaA reveals an open conformation characterized by an accessible cleft between the N- and C-terminal domains. Despite the conservation of residues involved in binding the allosteric activator UDP-GlcNAc, the enzyme is not strictly regulated by the substrate. Unlike S. aureus MnaA, the activity of P. alvei MnaA remains unaffected by tunicamycin.https://www.frontiersin.org/articles/10.3389/fmolb.2024.1470989/fullglycobiologykinetic constantsnon-hydrolyzing epimerasePaenibacillus alveiS-layer anchoringstructure-activity relationship |
| spellingShingle | Cordula Stefanović Max S. G. Legg Nick Mateyko Jakob J. Ender Tea Kuvek Chris Oostenbrink Christina Schäffer Stephen V. Evans Fiona F. Hager-Mair Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei Frontiers in Molecular Biosciences glycobiology kinetic constants non-hydrolyzing epimerase Paenibacillus alvei S-layer anchoring structure-activity relationship |
| title | Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei |
| title_full | Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei |
| title_fullStr | Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei |
| title_full_unstemmed | Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei |
| title_short | Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei |
| title_sort | insights into structure and activity of a udp glcnac 2 epimerase involved in secondary cell wall polymer biosynthesis in paenibacillus alvei |
| topic | glycobiology kinetic constants non-hydrolyzing epimerase Paenibacillus alvei S-layer anchoring structure-activity relationship |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2024.1470989/full |
| work_keys_str_mv | AT cordulastefanovic insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT maxsglegg insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT nickmateyko insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT jakobjender insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT teakuvek insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT chrisoostenbrink insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT christinaschaffer insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT stephenvevans insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei AT fionafhagermair insightsintostructureandactivityofaudpglcnac2epimeraseinvolvedinsecondarycellwallpolymerbiosynthesisinpaenibacillusalvei |