AUTHOR=Stefanović Cordula , Legg Max S. G. , Mateyko Nick , Ender Jakob J. , Kuvek Tea , Oostenbrink Chris , Schäffer Christina , Evans Stephen V. , Hager-Mair Fiona F. 

TITLE=Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=11

YEAR=2024

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2024.1470989

DOI=10.3389/fmolb.2024.1470989

ISSN=2296-889X

ABSTRACT=<sec><title>Introduction</title><p>S-layer anchoring in <italic>Paenibacillus alvei</italic> 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.</p></sec><sec><title>Methods</title><p>To better understand SCWP biosynthesis and identify strategies for inhibiting pathogens with comparable cell wall architecture, like <italic>Bacillus anthracis</italic>, MnaA and rational variants were produced in <italic>E. coli</italic> 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 <italic>in vitro</italic> supported by molecular docking experiments. Additionally, wild-type MnaA was crystallized.</p></sec><sec><title>Results</title><p>We present the crystal structure of unliganded <italic>P. alvei</italic> 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 <italic>K</italic><sub>M</sub> and <italic>k</italic><sub>cat</sub> of MnaA were determined to be 3.91 mM and 33.44 s<sup>-1</sup> for the forward, and 2.41 mM and 6.02 s<sup>-1</sup> 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 <italic>P. alvei</italic> 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 <italic>P. alvei</italic> MnaA and <italic>Staphylococcus aureus</italic> MnaA, which is inhibited by tunicamycin, revealed different residues exposed to the antibiotic excluding, those at the predicted allosteric site of <italic>P. alvei</italic> MnaA, corroborating tunicamycin resistance.</p></sec><sec><title>Conclusion</title><p>The unliganded crystal structure of <italic>P. alvei</italic> 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 <italic>S. aureus</italic> MnaA, the activity of <italic>P. alvei</italic> MnaA remains unaffected by tunicamycin.</p></sec>