The final, formatted version of the article will be published soon.
ORIGINAL RESEARCH article
Front. Mol. Biosci.
Sec. Glycoscience
Volume 11 - 2024 |
doi: 10.3389/fmolb.2024.1470989
Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei
Provisionally accepted- 1 Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Vienna, Austria
- 2 Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada
- 3 Department of Material Sciences and Process Engineering, University of Natural Resources and Life Sciences Vienna, Vienna, Austria
- 4 Department of Material Sciences and Process Engineering,, University of Natural Resources and Life Sciences Vienna, Vienna, Austria
Introduction: S-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.To 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.Results: We 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 PaMnaA. 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.The 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.
Keywords: Glycobiology, Kinetic constants, non-hydrolyzing epimerase, Paenibacillus alvei, S-layer anchoring, structure-activity relationship, Tunicamycin, X-ray structure
Received: 26 Jul 2024; Accepted: 03 Sep 2024.
Copyright: © 2024 Stefanović, Legg, Mateyko, Ender, Kuvek, Oostenbrink, Schäffer, Evans and Hager-Mair. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Fiona F. Hager-Mair, Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Vienna, Austria
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.