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 journalArticle

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

Fingerprint

Elapidae
Phospholipases A2
Taiwan
Isoenzymes
Enteropeptidase
Secretory Phospholipase A2
Computer Graphics
Sulfonation
Mutagenesis
Reptiles
Venoms
Dichroism
Molecular mass
Enzymes
Computer graphics
Circular Dichroism
Site-Directed Mutagenesis
Bioactivity
Escherichia coli
Mass spectrometry

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Characterization of phospholipase A2 (PLA2) from Taiwan cobra : Isoenzymes and their site-directed mutants. / Pan, Fu Ming; Chao, Shu Chi; Wu, Shih Hsiung; Chang, Wen Chang; Chiou, Shyh Horng.

In: Biochemical and Biophysical Research Communications, Vol. 250, No. 1, 08.09.1998, p. 154-160.

Research output: Contribution to journalArticle

@article{3fb37aa2746d420fbc0b6b2fa01d7531,
title = "Characterization of phospholipase A2 (PLA2) from Taiwan cobra: Isoenzymes and their site-directed mutants",
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.",
author = "Pan, {Fu Ming} and Chao, {Shu Chi} and Wu, {Shih Hsiung} and Chang, {Wen Chang} and Chiou, {Shyh Horng}",
year = "1998",
month = "9",
day = "8",
doi = "10.1006/bbrc.1998.9194",
language = "English",
volume = "250",
pages = "154--160",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Elsevier B.V.",
number = "1",

}

TY - JOUR

T1 - Characterization of phospholipase A2 (PLA2) from Taiwan cobra

T2 - Isoenzymes and their site-directed mutants

AU - Pan, Fu Ming

AU - Chao, Shu Chi

AU - Wu, Shih Hsiung

AU - Chang, Wen Chang

AU - Chiou, Shyh Horng

PY - 1998/9/8

Y1 - 1998/9/8

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0032497171&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032497171&partnerID=8YFLogxK

U2 - 10.1006/bbrc.1998.9194

DO - 10.1006/bbrc.1998.9194

M3 - Article

C2 - 9735349

AN - SCOPUS:0032497171

VL - 250

SP - 154

EP - 160

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 1

ER -