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  • Interest in DGKs increased as it became


    Interest in DGKs increased as it became clear that not only are they important for lipid homeostasis, they serve to modulate the relative levels of diacylglycerol (DAG) and phosphatidic AMG-458 mg (PtdOH) that play critical roles in a variety of signaling pathways (Eichmann and Lass, 2015; Liu et al., 2013) including neurotransmission (Raben and Barber, 2017; Tu-Sekine et al., 2015; Tu-Sekine and Raben, 2011). This highlights the need to understand the structure, regulation and catalytic mechanism of these enzymes. Indeed, lacking of the molecular and structural insight into DGKs has been a huge barrier for designing highly specific inhibitors for the DGKs to probe further roles and potential therapeutic strategies where possible.
    Overall structural features of lipid kinases Despite the differences in lipid substrates, an overall structure of lipid kinases must contain: a region/residue(s) for membrane association/stabilization, a domain to bind and orient the phosphor donor (ATP/CTP), and a domain for the binding and orientation of the phosphoryl acceptor (lipid substrate). These domains are oriented in a manner to permit the catalytic reaction (Cheek et al., 2002; Fabbro et al., 2015). While we have gained valuable insights into the nucleotide binding domains, the structure of the lipid substrate binding is not well understood. Understanding the architecture of these domains and their relationship to each other in each lipid kinase will provide valuable insights into the catalytic mechanism(s) of these enzymes.
    Prokaryotic DAG kinases There are two classes of prokaryotic DGKs designated as DGKA and DGKB (Van Horn and Sanders, 2012). Perhaps the most well-understood, and smallest, of these are the DGKAs. There are two types of DGKAs; one found in gram-positive bacteria and the other found in gram-negative bacteria. A hallmark of these DGKs are that they exist as homotrimeric proteins where each monomer contains three transmembrane-spanning regions (Li et al., 2013; Oxenoid et al., 2002). Interestingly, there are some important substrate and mechanistic differences between these two DGKAs. The one found in gram-positive bacteria prefers undecaprenol as a substrate while the gram-negative enzyme prefers diacylglycerol as a substrate although it also phosphorylates monoacylglycerol, as well as ceramide (Jerga et al., 2007). Mechanistically, it is interesting to note that this DGKA also show hydrolytic (ATPase) activity (Li et al., 2015). The prokaryotic DgkB from Staphylococcus aureus is essential for lipoteichoic acid synthesis (Matsuoka et al., 2011) and its solubility makes it particularly interesting with respect to its relevance to mammalian DGKs. Importantly, this is the first bona fide DGK with a solved structure (Miller et al., 2008). While the primary sequence similarity of DgkB compared to their eukaryotic cousins is low (15%–18%), the catalytic core is conserved (Jerga et al., 2007; Miller et al., 2008). DgkB consists of two domains: D1 and D2 (Fig. 1A), where both domains maintain an αβ fold. The D1 domain contains highly conserved ATP binding residues (Fig. 1A and C) and the D2 domain has a highly conserved glutamate (Glu 273), which likely serves as a catalytic base. The active site has been found located in the interdomain cleft but the lipid substrate binding site is still unclear. Another interesting point of DgkB structure is although each DgkB monomer is presumably supports catalysis independently, DgkB exist as extended dimers in solution as revealed by sedimentation velocity experiments (Miller et al., 2008). The established dimer interface is formed through the N-terminal domain (NTD-NTD) contacts. This interaction might play an important role by placing three conserved, positively charged surface residues near the negatively charged membrane so as to facilitate the extraction of the lipid substrate (Miller et al., 2008). There are two features of this enzyme that resemble the mammalian DGKs. First, there appears to be a conserved Asp-water-Mg2+ complex in the active site, although a second Mg2+ is essential to maintain a competent structure. Second, as noted above, the sequence of the catalytic site is conserved between DGKB and mammalian DGKs suggesting similar catalytic chemistries.