Glycoconjugates represent a class of macromolecules relevant for bacterial viability and virulence, as they play a key role in host adherence. Despite the capability to synthesize a diverse set of glycoconjugates, bacteria share common mechanisms of biosynthesis. The pathways involve transfer of a sugar molecule from a nucleotide activated sugar donor to a lipid acceptor polyprenol phosphate (Pren-P). The glycan-based macromolecules are diversified through the action of glycosyltransferases, which vary in their donor-molecule selectivity. Understanding the factors influencing selectivity of glycosyltransferases lays the basis for the development of novel selective antibiotics. A majority of GTs in the pgl operon of C. jejuni, C. concisus, C. fetus, and other Campylobacter members, namely PglA, PglJ, and PglH, belong to the GT-B superfamily in a subclass of membrane-associated (“monotopic”) proteins. The GT-B type fold is characterized by two β/α/β Rossmann-fold domains with the catalytic center located in a large cleft between them. In this work we report the first structural characterization of the glycosyltransferase PglA, from Campylobacter concisus ATCC 33237, as well as identification of its preferred substrate via nano differential scanning fluorimetry (nanoDSF) screening. Using X-ray crystallography, we have determined a 1.9 Å structure of PglA, bound to its native donor substrate, uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc). Guided by this structural information we designed a series of site-directed variants to gain understanding of the factors influencing PglA substrate specificity. The substrate of the next enzyme in the pathway, PglJ, has been identified as a 217 Da sugar in the literature, based on its mass spectrometry fragmentation pattern, presumably N-acetylhexosaminuronic acid of unknown stereochemistry. Herein, we report nanoDSF and luminescence based activity assay results showing that the C. concisus ATCC 33237 PglJ, reacts preferentially with UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) over its epimer, UDP-N-acetylglucosaminuronic acid (UDP-GlcNAcA).

Structure and specificity of the GT-B glycosyltransferases from Campylobacter concisus

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