Stefanović Cordula, Legg Max S G, Mateyko Nick, Ender Jakob J, Kuvek Tea, Oostenbrink Chris, Schäffer Christina, Evans Stephen V, Hager-Mair Fiona F
Department of Chemistry, Institute of Biochemistry, NanoGlycobiology Research Group, Universität für Bodenkultur Wien, Vienna, Austria.
Department of Biochemistry & Microbiology, University of Victoria, Victoria, BC, Canada.
Front Mol Biosci. 2024 Sep 26;11:1470989. doi: 10.3389/fmolb.2024.1470989. eCollection 2024.
S-layer anchoring in 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 , MnaA and rational variants were produced in 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 supported by molecular docking experiments. Additionally, wild-type MnaA was crystallized.
We present the crystal structure of unliganded 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 and of MnaA were determined to be 3.91 mM and 33.44 s for the forward, and 2.41 mM and 6.02 s 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 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 MnaA and MnaA, which is inhibited by tunicamycin, revealed different residues exposed to the antibiotic excluding, those at the predicted allosteric site of MnaA, corroborating tunicamycin resistance.
The unliganded crystal structure of 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 MnaA, the activity of MnaA remains unaffected by tunicamycin.
S层在[具体细菌名称未给出]中的锚定是通过S层同源结构域三聚体与次生细胞壁聚合物（SCWP）之间的非共价相互作用实现的，这确保了细菌细胞壁的结构完整性。在SCWP重复序列中，丙酮酸化的甘露糖胺作为配体，UDP-GlcNAc-2-表异构酶MnaA为SCWP生物合成提供UDP-甘露糖胺。
为了更好地理解SCWP生物合成并确定抑制具有类似细胞壁结构的病原体（如[具体细菌名称未给出]）的策略，在[具体表达系统未给出]中产生了MnaA及其合理变体，并测定了它们的动力学常数。在分子对接实验的支持下，测试了UDP-GlcNAc作为预测的别构激活剂和衣霉素作为MnaA潜在抑制剂的作用。此外，对野生型MnaA进行了结晶。
我们展示了在2.20 Å分辨率下解析的未结合配体的[具体细菌名称未给出]MnaA的晶体结构。它采用与其他细菌非水解UDP-GlcNAc 2-表异构酶一致的GT-B折叠。氨基酸序列比较揭示了MnaA中假定的和已知的催化及别构位点残基的保守性，这通过对Q42A、Q69A、E135A和H241A MnaA变体的分析得到证实。MnaA的正向反应动力学参数Km和kcat分别确定为3.91 mM和33.44 s，反向反应的Km和kcat分别为2.41 mM和6.02 s。虽然已提出UDP-GlcNAc的别构调节作为酶激活的机制，但未发现UDP-GlcNAc对[具体细菌名称未给出]MnaA的UDP-甘露糖胺表异构化至关重要。然而，添加5%的UDP-GlcNAc后反应速率加倍。出乎意料的是，UDP-GlcNAc类似物衣霉素不抑制MnaA。比较衣霉素与[具体细菌名称未给出]MnaA和受衣霉素抑制的[另一细菌名称未给出]MnaA结合的分子对接实验揭示了暴露于抗生素的不同残基，排除了[具体细菌名称未给出]MnaA预测别构位点处的残基，证实了对衣霉素的抗性。
[具体细菌名称未给出]MnaA的未结合配体晶体结构揭示了一种开放构象，其特征是在N端和C端结构域之间有一个可及的裂隙。尽管参与结合别构激活剂UDP-GlcNAc的残基保守，但该酶不受底物严格调节。与[另一细菌名称未给出]MnaA不同，[具体细菌名称未给出]MnaA的活性不受衣霉素影响。
