Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases

Abstract

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity.

Mining the Moraxella catarrhalis Genome: Identification of Potential Vaccine Antigens Expressed during Human Infection

“…However, there are numerous examples of enzymes that are located in the outer membrane of gram-negative bacteria, including a phosphomonoesterase (P4) and a glycerol-3-phosphodiester phosphodiesterase (protein D) in H. influenzae, an enolase in Pseudomonas aeruginosa, the lipid kinase YegS in Salmonella, and the lipid A acylase PagP and deacylase PagL found in Pseudomonas, Bordetella, and Salmonella (24,32,39,41,42,49). Additionally, the M. catarrhalis BRO-1 ␤-lactamase is in the outer membrane (12).…”

Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD–YeaZ–YjeE complex

“…29,30 It was further suggested that the different conformational states of these two dimeric complexes could form the basis for a switching mechanism between partner proteins. StYeaZ has been reported to act as a resuscitation promoting factor (RPF) in Salmonellae, 31 Our studies are aimed at the characterization of the structure and biochemical properties of essential proteins of unknown function in Salmonella typhimurium that might represent novel drug targets, including YeaZ, 28 YegS, 33 and YcbL. 34 New drugs are urgently required as the continual rise of high level resistance to current anti-bacterials presents increasing threats to human and animal health.…”

Signaling at the membrane interface by the DGK/SK enzyme family

“…One is the DgkB structure mentioned above. The second is that of a Salmonella protein of undetermined function, YegS (32). Comparison of these structures has yielded insight into potential substrate-binding regions, sites of regulatory lipid(s) binding, sites of membrane binding, and conformational changes that could modulate activity.…”

“…The YegS structure contains a hydrophobic region on the surface of Domain 1 that faces away from the substrate binding cleft (32). This region could represent a site for binding of lipids responsible for allosteric activation.…”