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Updated: Mar 25, 2026
Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression,
S I Bakholdina1, N M Tischenko, E V Sidorin
1Elyakov Pacific Institute of Bioorganic Chemistry, Russian Academy of Sciences, Far East Branch, Vladivostok, 690022, Russia. sibakh@mail.ru.
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.
Insights
This study details the cloning and characterization of phospholipase A1 (PlaA) from Yersinia pseudotuberculosis. The refolded enzyme shows broad pH and temperature activity, with structural insights revealing differences from E. coli PlaA.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- The pldA gene encodes the membrane-bound phospholipase A1 in Yersinia pseudotuberculosis.
- Understanding this enzyme is crucial for its potential applications and for comparative studies.
Purpose of the Study:
- To clone and express the pldA gene from Y. pseudotuberculosis in E. coli.
- To isolate, purify, refold, and characterize the recombinant phospholipase A1 (rPlaA).
- To model the enzyme's structure and identify key functional residues.
Main Methods:
- Gene cloning and expression in E. coli.
- Protein purification using ion-exchange and gel-filtration chromatography.
- Mass spectrometry (MALDI-TOF MS) for molecular mass determination.
- Enzyme refolding and activity assays.
- Bioinformatic tools for structural modeling.
Main Results:
- Recombinant PlaA (rPlaA) was successfully expressed and purified as an inactive monomer.
- Refolded rPlaA demonstrated hydrolytic activity against a phosphatidylcholine substrate in the presence of calcium ions.
- The enzyme exhibited optimal activity at 37°C and a wide pH range (4-11), with maximum activity at pH 10.
- Structural modeling revealed potential differences between Y. pseudotuberculosis and E. coli PlaA, impacting function.
Conclusions:
- The study successfully characterized the functional properties of Y. pseudotuberculosis phospholipase A1.
- Structural insights provide a basis for understanding enzyme-substrate interactions and inter-species differences.
- The findings contribute to the knowledge of bacterial phospholipases and their potential roles.
