PlsY

PlsY (PDB ID: 5XJ5) from Aquifex aeolicus

Created by: Grace Nahorney

Integral membrane protein, glycerol 3-phosphate (G3P) acyltransferase PlsY (PDB ID: 5XJ5) is a metabolic enzyme found in bacterial Aquifex aeolicus (1). PlsY is involved in the pathway of phospholipid metabolism, acting as a catalyst in the monoacylglycerol form. PlsY catalyzes the step to form lysophosphatidic acid (lysoPA) by the acylation of G3P in bacterial phospholipid biosynthesis (2). LysoPA is a water soluble phospholipid and a signaling molecule that undergoes acylation twice to form phosphatidic acid (PA). If lysoPA is not released, it acts as a product inhibitor and interacts with G3P binding residues by forming three hydrogen bonds with the Thr-41 hydroxyl group and the backbone amides of Ala-40 and Thr-41 (3). The release of the LysoPA is driven by the substrates being present in high concentrations. PA is an important intermediate in phospholipid metabolism. Therefore, PlsY is critical in enabling bacterial phospholipid biosynthesis (2,4).

Bacteria have two catalytic G3P acyltransferases, PlsY and PlsB, that catalyze the formation of lysoPA. PlsB has eukaryotic homologs and is responsible for bacterial growth using an acyl donor called acyl-CoA. PlsY lacks eukaryotic homologs and has a fatty acyl donor called acyl-phosphate (acylP) (2, 5). PlsY is found in many gram-positive bacteria including Enterococcus faecium and Streptococcus pneumoniae. These two pathogens have the highest multi-drug resistance according to the World Health Organization, making PlsY a subject of interest in drug discovery (2). PlsX (PDB ID: 6A1K) is an acylP synthesizing enzyme. PlsX paired with PlsY catalyzes a two-step pathway to form membrane phospholipids in Streptococcus pneumoniae in Bacillus subtilis cells (5). PlsX and PlsY in B. subtilis cells assist the activity of PlsB in E. coli. The PlsX/Y pathway is the most widely distributed pathway for the initiation of phospholipid synthesis by forming phosphatidic acid in bacteria. This pathway generates a unique acyl-phosphate intermediate via PlsX that can then be used as a substrate for PlsY. The essential enzymes, PlsX and PlsY, are unique to bacteria and thus represent appealing targets for the development of antibacterial agents to combat pathogenic resistance to drug therapies. The mutation of either PlsY or PlsX is lethal, suggesting that both enzymes are essential for G3P transferase activity (5, 6).

PlsY from Aquifex aeolicus was expressed in Escherichia coli (E. Coli) BL21 using the lipidic cubic phase method. Crystallization was performed under the conditions of 7.8 monoacylglycerol (7.8 MAG), 0.1 M ammonium sulfate, 25-30% triethylene glycol, and 0.1 M glycine/HCl at a pH of 3.8 and a temperature of 293.0 K. The ligand ethylene glycol was only used to induce crystallization and x-ray diffraction was used to obtain the structural data for PlsY (2).

The Expasy database determined that PlsY has a molecular weight of 21844.09 Da and an isoelectric point of 10.16 (7). The entire protein was able to be sequenced. The primary structure of PlsY consists of 201 residues. The secondary structure includes alpha helices and random coils. PlsY does not contain beta sheets. There are twelve alpha helices composed of 148 residues and seven of the helices are transmembrane helices (TMHs). The structure is 73% helical. Random coils are present to an unknown degree. The amino terminus and two short cytoplasmic loops of PlsY are located on the external face of the membrane. There are 6 loops with conserved amino acids connecting the alpha helices (2). The hydrophobic effect induces tertiary structure. In a process called hydrophobic collapse, the hydrophobic residues cluster on the interior of the protein causing it to fold into a compact structure. The collapse decreases the surface-to-volume ratio of the protein, minimizing its interaction with the surroundings (8). PlsY lacks quaternary structure as it is made of only 1 subunit (1). This single subunit structure interacts with PlsX (6).

PSI-BLAST is used to find proteins with similar primary structures to a protein query. The proteins with similar primary structures are called subjects. The E value is determined by comparing the sequences of proteins to find amino acids present in the subject protein and absent in the query protein. An E value below 0.05 indicates a high similarity between proteins (9). The PSI-BLAST of PlsY resulted in no significant proteins having a similar primary structure. The Dali server compares the tertiary structures of proteins and uses a sum-of-pairs method to produce a measure of similarity between proteins. Proteins with similar folds have a Z score above 2.0 (10).

Endophilin-A1 (PDB ID: 1ZWW) and PlsY are both transferases, but Endophilin-A1 is found in Mus musculus (11). Endophilin-A1 functions in endocytosis of clathrin-coated vesicles, signal trafficking, and apoptosis. Endocytosis is mediated by the levels of calcium in the cell (12). Endophilin-A1 did not give an E value in PSI-BLAST because its primary structure is not sufficiently similar to PlsY. The Dali server gave a Z score of 6.7, indicating that Endophilin-A1 and PlsY have a similar tertiary structure (10). Endophilin-A1 is 512 residues long with 2 chains that are 256 residues each. It is longer than PlsY by 311 residues. The secondary structures of both transferases contain alpha helices and lack beta sheets. Endophilin-A1 is 66% helical and contains 7 alpha helices made of 170 residues. PlsY has 5 more alpha helices than Endophilin-A1 (1, 11). Endophilin-A1 has one ligand known as the cadmium ion (11). The cadmium ion is bound to the dimer and can coordinate with calcium ions. The cadmium ligand enables the regulation of intracellular calcium levels thus allowing clathrin-coated vesicles to be endocytosed (12).

PlsY has a cone-like structure, whereas Endophilin-A1 forms a crescent-shaped dimer with a group of three and a group of four alpha helices that come together. Endophilin-A1 has important residues that help support its structure. The dimer is stabilized by salt-bridges formed between Lys-39 and Asp-106. Asp-106 also interacts with the cadmium ion making Asp-106 essential in regulating calcium. PlsY uses glutamic acid residues for stabilization (2). Arg-45 and Glu-103 form hydrogen bonds with the hydroxyl group of Tyr-57. The binding sites on Endophilin-A1 are the cadmium ions (12). Endophilin-A1 and PlsY do not share common functionalities, but they do share similar folds.

PlsY has a v-shaped active site formed by the orientation of the TMHs. The (2S)-2,3-dihydroxypropyl(7Z)-pentadec-7-enoate. A ligand pocket is formed by the folding of PlsY and can interact with the glycine ligand. As PlsY is folded into its tertiary structure, ligand pockets are formed where ligands can interact with amino acids. An example of this interaction is glycine hydrogen bonding to Ser-73 and Gly-324. Glycine will bind a phosphate when attached to either residue. The sulfate ion hydrogen bonds to the G3P binding pocket containing Asn-180, Lys-104, Arg-45, and Ser-35. It also can attach either one or two oxygen atoms to create a bridge that increases the stabilization of the active site. (2S)-2,3-dihydroxypropyl(7Z)-pentadec-7-enoate hydrogen bonds to Ile-131, Leu-133, and Trp-134 (1, 2). The sulfate ion and (2S)-2,3-dihydroxypropyl(7Z)-pentadec-7-enoate can bind to multiple ligand pockets, but glycine is found in only one ligand pocket. The interaction of glycine with PlsY is more specific than with the sulfate ion or (2S)-2,3-dihydroxypropyl(7Z)-pentadec-7-enoate (2).

There are 3 regions on the cytoplasmic surface with fully conserved amino acid sequences. Upon site-directed mutagenesis, it was determined that these three regions are crucial for catalysis (5). Region 1 (residues 35-46) is found on the first cytoplasmic loop. It contains a serine and arginine residue. If the essential residues, Ser-35 and Arg-45, are mutated, almost no PlsY activity remains. The side chains of His-92 and Thr-41 contribute to acylP binding. Region 2 (residues 100-177) is found on the second cytoplasmic loop. Substitution of glycine to alanine caused a K_m defect for G3P binding. This defect lead to the conclusion that Region 2 corresponds to the G3P binding site. If glycine is mutated by the addition of a methyl side chain, then most acyltransferase activity is inactivated. His-177 is a critical residue for G3P binding as it interacts with the hydroxyl group on G3P (2). Region 3 (residues 185-197) contains conserved residues, His-185 and Asn-190, that are essential for catalytic activity. A glutamic acid residue is critical to maintaining the architectural structure of PlsY (2, 5).

The substrates are generated on the cytoplasmic membrane. The two substrates are G3P and palmitoyl phosphate (16: 0-P). The G3P binding pocket binds a phosphate through the glycine ligand. The G3P binding pocket is sensitive to missense mutations (2). Alanine mutations cause over 92% loss of activity and a 5 to 36 fold increase in G3P K_m. Arg-45 is required for G3P to bind. Arginine substituted by lysine causes a 90% loss of activity and a 14 fold increase in G3P K_m. Lys-104 mutated to arginine causes a 25% loss of activity and has a mild effect on G3P K_m. His-177 is essential and will result in loss of all function of the binding pocket if mutated. The residues in the G3P binding pocket are highly conserved (5). Deep in the membrane there is a hydrophilic phosphate hole that secures the phosphate of 16: 0-P. Three hydrogen bonds are formed between the carbonyl oxygen on 16: 0-P and one hydroxyl group of Thr-41 and two backbone amides on Ala-40 and Thr-41. The amine of Lys-104 also has a charge-charge interaction with G3P in the phosphate hole. TMH2 and TMH4-6 form a hydrophobic groove that holds the 16: 0-P. 16: 0-P binds to the glycine on the second alpha helix and to the side chain of TMH4. The alpha helix acquires a positive dipole on the N terminus and a negative dipole at the C terminus. The partial positive charge neutralizes the phosphate’s charge on 16: 0-P (2, 5).

Ultimately, the biological significance of PlsY is its ability to synthesize phospholipids through acyltransferase activity. PlsY catalyzes the transfer of an acyl group from acyl-phosphate (acyl-PO₄) to glycerol 3-phosphate (G3P) to first form LysoPA and then PA (1, 2). Antimicrobial inhibitors are being designed for PlsY to destroy pathogenic bacteria by disabling the synthesis of the phospholipid membrane. Inhibitors for PlsY have been identified to be used in the gram-positive bacteria Staphylococcus aureus. Staphylococcus aureus is found on the skin and in the respiratory tract as it is responsible for skin infections and food poisoning (13). The ability of an inhibitor to disable PlsY stops the spread of the infection commonly known as “staph.” Cells that cannot actively regulate molecules that cross the membrane due to the membrane’s disruption will die. PlsY is an important target for antibacterial therapy development. Furthermore, the understanding of the multistep PlsX/Y pathway can be used to create antibodies of multi-drug resistant bacteria that disable or inhibit the synthesis of the phospholipid bilayer thus causing a cell’s death.