Åke Wieslander

WaaG is a glycosyltransferase that is involved in the biosynthesis of lipopolysaccharide in Gram-negative bacteria. Inhibitors of WaaG are highly sought after as they could be used to inhibit the biosynthesis of the core region of lipopolysaccharide, which would improve the uptake of antibiotics. Herein, we establish an activity assay for WaaG using ¹⁴C-labeled UDP-glucose and LPS purified from a ∆waaG strain of Escherichia coli. We noted that addition of the lipids phosphatidylglycerol (PG) and cardiolipin (CL), as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) increased activity. We then use the assay to determine if three molecular scaffolds, which bind to WaaG, could inhibit its activity in vitro. We show that 4-(2-amino-1,3-thiazol-4-yl)phenol inhibits WaaG (IC₅₀ 1.0 mM), but that the other scaffolds do not. This study represents an important step towards an inhibitor of WaaG by fragment-based lead discovery.

The human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid‐detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT‐A fold, similar to an established structure, and belong to CAZy GT‐family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDP‐galactose but also UDP‐glucose as donors, making mono‐, di‐ and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major β1,6GlcβGalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function.

We have used multivariate statistics and z-scales to represent peptide sequences in a PLS-QSAR model of previously studied binding affinities [Kofler,M., Motzny,K. and Freund,C. (2005b)Mol. Cell. Proteomics, 4, 1797–1811.] of two GYF domains to an array of immobilized synthetic peptides. As a result, we established structural determinants of the binding specificities of the two proteins. Our model was used to define new sets of yeast proteins potentially interacting with Syh1 (YPL105C) and Smy2 (YBR172C). These sets were subsequently examined for co-occurrence of Gene Ontology terms, leading to suggest a group of likely interacting proteins with a common function in mRNA catabolism. Finally, subcellular localization of a GFP-fused Syh1 and Smy2 reinforced the possibility that these proteins reside in cytoplasmic sites of mRNA degradation, thereby providing experimental confirmation to the predictions from the model.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer‐ and nonbilayer‐prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress (‘spontaneous curvature’). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene‐lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer‐prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid‐crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis‐pyrene‐lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.