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 Km 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 Km. 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 Km. Lys-104 mutated
to arginine causes a 25% loss of activity and has a mild effect on G3P Km.
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.