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Instruments / Technical Area	Publication Details	Published	Actions
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography Krios II-Titan Krios II at Diamond	Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide Audrey Le Bas , Bradley R. Clarke , Tanisha Teelucksingh , Micah Lee , Kamel El Omari , Andrew M. Giltrap , Stephen A. Mcmahon , Hui Liu , John H. Beale , Vitaliy Mykhaylyk , Ramona Duman , Neil G. Paterson , Philip N. Ward , Peter J. Harrison , Miriam Weckener , Els Pardon , Jan Steyaert , Huanting Liu , Andrew Quigley , Benjamin G. Davis , Armin Wagner , Chris Whitfield , James H. Naismith Open Biology 15 DOI: 10.1098/rsob.240310 Diamond Proposal Number(s): [33941] Open Access Show Abstract ▼ Abstract: The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.	Jan 2025
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria Andrew Bell , Emmanuele Severi , Micah O. Lee , Serena Monaco , Dimitrios Latousakis , Jesus Angulo , Gavin H. Thomas , James Naismith , Nathalie Juge Journal Of Biological Chemistry DOI: 10.1074/jbc.RA120.014454 Open Access Show Abstract ▼ Abstract: The human gut symbiont Ruminococcus gnavus scavenges host‐derived N‐acetylneuraminic acid (Neu5Ac) from mucins, by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D nuclear magnetic resonance (NMR), we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-DANA intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram negative and Gram positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionisation spray mass spectrometry (ESI-MS) that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolise 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry (DSF) analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli was dependent on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.	Jul 2020

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