School of Chemistry - Research Publications

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Author: Williams, Spencer × Start date: 2020 × End date: 2024 × Type: Preprint ×

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    Defining the molecular architecture, metal dependence, and distribution of metal-dependent class II sulfofructose-1-phosphate aldolases
    Sharma, M ; Kaur, A ; Madiedo Soler, N ; Lingford, J ; Epa, R ; Goddard-Borger, E ; Davies, G ; Williams, S ( 2023-08-08)
    Sulfoquinovose (SQ or 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for the breakdown of SQ is the sulfoglycolytic Embden-Meyerhof-Parnas (sulfo-EMP) pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is well studied for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures in complex with the substrate SFP and product DHAP reveal a dimer-of-dimers (tetrameric) assembly, and identify the sulfonate binding pocket that defines the substrate specificity of these enzymes, two metal binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfo-EMP gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases occur exclusively within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.
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    Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target
    Sobala, ŁF ; Fernandes, PZ ; Hakki, Z ; Thompson, AJ ; Howe, JD ; Hill, M ; Zitzmann, N ; Davies, S ; Stamataki, Z ; Butters, TD ; Alonzi, DS ; Williams, SJ ; Davies, GJ ( 2020-07-01)
    ABSTRACT Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the soleendo-acting glycoside hydrolase involved in N-glycan trimming and unusually is located within the Golgi, where it allows ER escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur upon substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. The structures inspired the development of new inhibitors that disrupted host protein N-glycan processing of viral glycans and reduced infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts of developing broad-spectrum antiviral agents and bring about a more detailed view of the biology of mammalian glycosylation. SIGNIFICANCE STATEMENT The glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play a key role in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, and one with great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase, MANEA, provides an alternative processing pathway to evade glucosidase inhibitors. We report the 3D structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.