Characterization of phospholipase A2 (PLA2) from Taiwan cobra: Isoenzymes and their site-directed mutants

Fu Ming Pan, Shu Chi Chao, Shih Hsiung Wu, Wen Chang Chang, Shyh Horng Chiou

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

Extracellular and secretory phospholipase A2 (PLA2), a class of phospholipid digesting enzyme, is widely distributed in animal venoms of reptiles and insects. Two cDNAs encoding PLA2 isoenzymes from Taiwan Cobra (Naja naja atra) were cloned into pQE-30 plasmid vector and expressed in Escherichia coli. The recombinant products were subjected to refolding using sulfonation under reduction/oxidation conditions with glutathione and enterokinase removal of His-tag, resulting in the active recombinant PLA2 with the same molecular masses of native enzymes as determined by mass spectrometry. The recombinant PLA2 was also shown by circular dichroism to possess a secondary structure similar to native PLA2. The enzymatic activity of the major isoenzyme (PLA2-1) is higher than the other minor isoenzyme (PLA2-2), which shows two amino acid difference from PLA2-1. Site-directed mutagenesis was used to probe the structure/function relationship of two highly conserved residues among all reported PLA2, i.e., His-47 and Asp-93. Replacement of His-47 residue by either Ala or Arg resulted in the complete loss of activity. Similarly, the mutant Asp-93→Asn (D93N) also retained little activity. These results suggest that both His-47 and Asp-93 are essential for the catalytic activity of PLA2. Computer graphic study, based on homology modelling, highlights the differences between native PLA2 isoenzymes and their site-directed mutants, which may account for the differences in the observed biological activity.

Original languageEnglish
Pages (from-to)154-160
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume250
Issue number1
DOIs
Publication statusPublished - Sep 8 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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