From Protease to Decarboxylase: THE MOLECULAR METAMORPHOSIS OF PHOSPHATIDYLSERINE DECARBOXYLASE

Abstract

Phosphatidylserine decarboxylase (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylase containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is formed in a concerted reaction when the PSD proenzyme undergoes an endoproteolytic cleavage into a large β-subunit, and a smaller α-subunit, which harbors the prosthetic group at its N terminus. The mechanism of PSD proenzyme cleavage has long been unclear. Using a coupled in vitro transcription/translation system with the soluble Plasmodium knowlesi enzyme (PkPSD), we demonstrate that the post-translational processing is inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride. Comparison of PSD sequences across multiple phyla reveals a uniquely conserved aspartic acid within an FFXRX6RX12PXD motif, two uniquely conserved histidine residues within a PXXYHXXHXP motif, and a uniquely conserved serine residue within a GS(S/T) motif, suggesting that PSDs belong to the D-H-S serine protease family. The function of the conserved D-H-S residues was probed using site-directed mutagenesis of PkPSD. The results from these mutagenesis experiments reveal that Asp-139, His-198, and Ser-308 are all essential for endoproteolytic processing of PkPSD, which occurs in cis. In addition, within the GS(S/T) motif found in all PSDs, the Gly-307 residue is also essential, but the Ser/Thr-309 is non-essential. These results define the mechanism whereby PSDs begin their biochemical existence as proteases that execute one autoendoproteolytic cleavage reaction to give rise to a mature PSD harboring a pyruvoyl prosthetic group.

Keywords: Autoendoproteolysis; Membrane; Phosphatidylserine Decarboxylase; Phospholipid; Plasmodium; Protease; Pyruvoyl-enzyme.

FIGURE 1.

Phenylmethylsulfonyl fluoride inhibits processing of in vitro expressed His₆-Δ34PkPSD proenzyme. In vitro expression of His₆-Δ34PkPSD was conducted for 40 min at 30 °C using a TnT Quick-coupled transcription/translation system. After the first 20 min of the reaction, PMSF was added to final concentrations of 0, 0.5, 2.5, or 5 mm. At 40 min of incubation time, translation was arrested by the addition of 0.2 mm cycloheximide and the post-translational processing reaction continued for 40 min in the presence of 0.1 mg/ml of dioleoylphosphatidylserine liposomes. Upon the completion of the reactions, electrophoresis and Western blot analysis was conducted to detect the His₆ epitope present in both the nascent and mature His₆-Δ34PkPSD proteins. The percentages of proenzymes (PROENZ) and processed mature β-subunit (β-SUB) are shown below the blots. The protein band intensities of the proenzymes and processed β-subunits were quantified using ImageJ software. Data are mean ± S.D. for 3 experiments.

FIGURE 2.

Chimeric MBP-His₆-Δ34PkPSD is highly expressed and processed in E. coli. A, an N-terminal MBP fused to His₆-Δ34PkPSD was expressed in a Rosetta E. coli strain by 0.3 mm IPTG induction for 20 min, 40 min, or 2 h, at 37 °C. Cells were harvested and 0.01 A₅₅₀ units were subjected to electrophoresis and Western blot analysis using an anti-His₆ antibody. Values shown under the blots quantify the percentage of proenzyme (PROENZ) and β-subunit (β-SUB) at each time point expressed as the mean ± range determined in two experiments. B, the α-subunit of the expressed PkPSD contains the pyruvoyl moiety. MBP-His₆-Δ34PkPSD was expressed in a Rosetta E. coli strain by IPTG induction for 2 h at 37 °C. Cell-free extracts were prepared, and PkPSD proteins were purified by amylose column affinity chromatography and MBP was removed by factor Xa treatment. The α- and β-subunits of His₆-Δ34PkPSD were separated on SDS-PAGE. Gel pieces containing α-subunits were proteolyzed and analyzed by mass spectrometry. The MS/MS fragmentation pattern of the Pyr-SIVVIFENK peptide of the α-subunit is shown.

FIGURE 3.

PMSF inhibits processing of the MBP-His₆-Δ34PkPSD proenzyme. A, experimental scheme to test in vitro processing of E. coli expressed MBP-His₆-Δ34PkPSD proenzyme. MBP-His₆-Δ34PkPSD was expressed by 0.3 mm IPTG induction for 20 min 37 °C. Cell-free extracts were prepared and then incubated in vitro with dioleoylphosphatidylserine (DOPS) for the indicated times to allow maturation of the PkPSD proenzymes. 10 mm PMSF was added to the in vitro reaction to inhibit processing. B, Western blot analysis of MBP-His₆-Δ34-PkPSD fusion proteins that were incubated for indicated time in the absence or presence of PMSF. C, the percentages of processed enzymes are shown. The protein band intensities of the proenzymes and processed β-subunits on the Western blot were quantified using ImageJ software. Data are mean ± S.E. for 6 experiments.

FIGURE 4.

PkPSD contains contextually conserved amino acids across diverse phyla. The alignment of the sequences of PSD proenzymes from E. coli, S. cerevisiae, A. thaliana, P. falciparum, P. knowlesi, M. musculus, and H. sapiens reveals one conserved aspartic acid residue (Asp-139 in PkPSD), and two conserved histidine residues (His-195 and His-198 in PkPSD), and a conserved serine residue (Ser*-308 in PkPSD) in all PSD enzymes. The conserved primary sequence context for these conserved residues is shown below the box. Upon proteolytic cleavage of the proenzyme, the Ser* residue is converted into a pyruvoyl prosthetic group.

FIGURE 5.

Asp-139 and His-198 are essential for PkPSD processing. A, the schematic shows the amino acid residues targeted in these experiments in black. Western blot analyses of wild type (WT) MBP-His₆-Δ34PkPSD fusion protein and proteins harboring H195A/H198A, H198A, H195A, D139A, and D139N mutations are shown. The proteins were expressed using a 2-h IPTG induction in Rosetta E. coli strains. The Mock condition is the bacterial strain without plasmid. The percentages of proenzymes (PROENZ) and the β-subunit of the processed enzymes (β-SUB) are shown. The protein band intensities of the proenzymes and processed β-subunits were detected with anti-His₆ antibody and quantified by ImageJ software. Data are mean ± S.E. for 3 experiments. B, PSD enzyme assays were performed with cell-free extracts from Rosetta E. coli strains expressing wild type and mutant enzymes described in A. Values are mean ± S.E. for 3 experiments each performed in duplicate. C, His-195 is not essential for PkPSD processing. After a 2-h IPTG induction, cells were harvested and re-suspended in fresh LB-glucose medium without IPTG. Tetracycline (10 μg/ml) was added to the culture medium to arrest translation. Cells were incubated for an additional hour at 37 ºC. Western blot analysis was conducted to detect the His₆ epitope. Percentages of proenzymes and processed enzymes are shown below the blots. Data are mean ± S.E. for 3 experiments. D, PSD enzyme assays were performed using the same samples from the reactions shown in panel C. Data are mean ± S.E. for 3 experiments each performed in duplicate.

FIGURE 6.

Gly-307 and Ser-308 are essential for MBP-His₆-Δ34PkPSD processing, but Ser-309 is non-essential. A, the schematic shows the amino acids targeted in these experiments in black. Western blot analyses of wild type MBP-His₆-Δ34PkPSD fusion protein and mutagenized proteins of G307A, G307P, S308A, S308T, S309A, and S309T are shown. The proteins were expressed for 2 h following IPTG induction from Rosetta E. coli strains harboring no vector (Mock), or plasmids encoding the designated mutant proteins. The percentages of proenzymes (PROENZ) and processed enzymes (β-SUB) are shown below the blot. Data are mean ± S.E. for 3 experiments. B, PSD enzyme assays were performed with cell free extracts from Rosetta strains expressing the designated mutant proteins. Data are mean ± S.E. for 3 experiments each performed in duplicate.

FIGURE 7.

MBP-His₆-Δ34PkPSD processing is a unimolecular reaction occurring in cis. Wild type MBP-Δ34PkPSD, lacking the His₆ epitope tag (0.1 μg/μl final protein concentration), was mixed with wild type MBP-His₆-Δ34PkPSD, or catalytically inactive mutant forms of MBP-His₆-Δ34PkPSD harboring H198A, H139A, or S308A substitutions (0.1 μg/μl final protein concentration). In panel A, the reaction mixture was incubated for 2 h at 30 °C. In panel B, the reaction mixture was incubated for 20 h at 25 °C. After the reaction period, samples were analyzed by Western blot analysis with anti-His₆ and anti-MBP antibodies as indicated. Results shown are from one of three experiments.

FIGURE 8.

Serine protease motifs and processing of PSD enzymes. A, the PSD proenzyme contains a D-H-S catalytic triad that is conformationally activated by PS. B, activation of Ser*-308 in PkPSD results in attack of the peptide bond between Gly-307 and Ser*-308, and formation of an acyl-enzyme intermediate. The acyl-enzyme intermediate is cleaved by general base catalysis by His-198 to produce α- and β-subunits. C, the initial form of the free α-subunit harbors a dehydroalanine (derived from Ser*-308) at its N terminus. The dehydroalanine undergoes an elimination reaction with loss of NH₃ to yield a pyruvoyl prosthetic group, which serves as the critical active site substituent for decarboxylating PS. The active site Ser*-308 for the protease reaction becomes the active site pyruvoyl (Pyr) prosthetic group for the decarboxylase reaction.