Galactolipids are characteristic lipids of the photosynthesis membranes of higher plants and cyanobacteria. Due to their close relationship to the stability of the photosystem protein complexes, the biogenesis of galactolipids has been intensively studied on the genetic and molecular levels. There are two major types of galactolipids in chloroplastic membranes: monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG). Under phosphate-limiting conditions, the amount of DGDG increases dramatically to allow for phosphate salvage from phospholipids. In Arabidopsis thaliana, the membrane-associated glycosyltransferase digalactosyldiacylglycerol synthase 2 (atDGD2) is highly responsive to phosphate starvation and is significantly upregulated during such conditions. The lipid galactosylation reactions are also fundamentally interesting as they require a catalyst that is capable of bringing a hydrophilic and lipophilic substrate together at the solution-membrane phase border. Here, we present the X-ray crystal structure of atDGD2, which is the first reported DGDG synthase structure. AtDGD2 is most structurally similar to functionally unrelated GT-B enzymes. Interestingly, in spite of significant donor substrate binding differences, we identified four amino acids (Gly22, His151, Lys243, and Glu321, atDGD2 numbering) which were entirely conserved between the structurally similar enzymes. We also investigated the membrane interaction kinetics and membrane anchoring mechanism of atDGD2. This demonstrated that atDGD2 is membrane-bound but also showed that membrane binding is highly dynamic. Furthermore, our structural information in context of previous biophysical studies highlights regions of the enzyme exhibiting a high degree of structural plasticity, which we propose to be important for allowing atDGD2 to quickly adapt its activity based on the membrane lipid environment.

Galactolipids are characteristic lipids of the photosynthetic membranes. They are highly enriched in the chloroplast and are present in photosystem structures. There are two major types of galactolipids, i.e., monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) in chloroplastic membranes, which amount to similar to 50 and similar to 20 mol % of the total chloroplast lipids, respectively. Under phosphate-limiting conditions, the amount of DGDG increases dramatically for rescuing phosphate from phospholipids. In Arabidopsis thaliana, the gene digalactosyldiacylglycerol synthase 2 (DGD2) encodes a membrane-associated glycosyltransferase. The gene expression is highly responsive to phosphate starvation and is significantly upregulated in this case. To understand the molecular mechanism of DGD2, we established a protocol for DGD2 expression and purification in an Escherichia coli-based system. The work involved optimization of the expression condition and the purification protocol and a careful selection of buffer additives. It was found that a removal of around 70 C-terminal residues was necessary to produce a homogeneous monomeric protein sample with high purity, which was highly active. The purified sample was characterized by an activity assay for enzyme kinetics in which a range of membrane mimetics with different lipid compositions were used. The results demonstrate that DGD2 activity is stimulated by the presence of negatively charged lipids, which highlight the importance of the membrane environment in modulating the enzyme's activity. The study also paves way for future biophysical and structural studies of the enzyme.

Monotopic glycosyltransferases (GTs) interact with membranes via electrostatic interactions. The N-terminal domain is permanently anchored to the membrane while the membrane interaction of the C-terminal domain is believed to be weaker so that it undergoes a functionally relevant conformational change upon donor or acceptor binding. Here, we studied the applicability of this model to the glycosyltransferase WaaG. WaaG is involved in the synthesis of lipopolysaccharides (LPS) in Gram-negative bacteria and was previously categorized as a monotopic GT. We analyzed the binding of WaaG to membranes by stopped-flow fluorescence and NMR diffusion experiments. We find that electrostatic interactions are required to bind WaaG to membranes while mere hydrophobic interactions are not sufficient. WaaG senses the membrane's surface charge density but there is no preferential binding to specific anionic lipids. However, the binding is weaker than expected for monotopic GTs but similar to peripheral GTs. Therefore, WaaG may be a peripheral GT and this could be of functional relevance in vivo since LPS synthesis occurs only when WaaG is membrane-bound. We could not observe a C-terminal domain movement under our experimental conditions.