Recombinant phospholipase A1 of the outer membrane of psychrotrophic Yersinia pseudotuberculosis: Expression, purification, and characterization

Abstract

The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy

“…Recombinant GFP in PBS has a CD spectrum with a maximum at 198 nm and only one minimum at 220 nm, characteristic of proteins with β-pleated sheet structure ( Figure 8 A). As shown earlier [ 30 ], the Y. pseudotuberculosis phospholipase A 1 , which, similar to GFP, has the cylindrical β-sheet structure, exhibits a CD spectrum with a positive band at 193 nm and a broad negative band centered at 215 nm. The CD spectra of the recombinant proteins PldA-GFP, PldA and GFP in SDS solutions are characterized by a maximum at 197 nm and two minima at 208–207 nm and 219–217 nm, and are typical for mixed α-β proteins.…”

Phosphatidylinositol 4,5-Bisphosphate-Dependent Oligomerization of the Pseudomonas aeruginosa Cytotoxin ExoU

“…The formation of oligomers is not unique to ExoU but is widespread among phospholipases. Crystallization studies have shown oligomerization to occur with both bacterial and eukaryotic phospholipases, including several PLA 2 enzymes found in snake venom (42)(43)(44), PldA of Yersinia pseudotuberculosis (45), human pancreatic prophospholipase A 2 (46), and calcium-independent cytosolic phospholipase A 2 (47). Our results suggest that localization to the host cell plasma membrane and, in particular, to PI(4,5)P 2 in the inner leaflet of the plasma membrane causes ExoU oligomerization.…”