Purification and characterization of the second Streptomyces phospholipase A2 refolded from an inclusion body.
A secreted phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber A-2688, previously identified by us, is the first PLA(2) identified in prokaryotes. Genome sequence data of Streptomyces coelicolor A3(2) indicates that the bacterium carries two genes encoding hypothetical PLA(2)s, which exhibit 100 and 78% identity, respectively, to the S. violaceoruber PLA(2). In this study, we named the former and latter proteins as the first and second PLA(2)s, respectively. When the second PLA(2) was expressed in Escherichia coli cells, it formed an inclusion body. The present study demonstrates a method to purify it to homogeneity without the disappearance of the enzymatic activity: the inclusion body was washed with sodium deoxycholate and dissolved in the presence of 2 M urea at pH 12, then refolded by the dilution method. The refolding of enzyme was confirmed by the circular dichroism spectrum. The second PLA(2) purified to homogeneity had the same specific activity as that of the S. violaceoruber PLA(2) and the yield was approximately 6.8 mg/L culture. The second PLA(2) exhibits similar enzymatic properties to the S. violaceoruber PLA(2), except that the former enzyme does not utilize phophatidic acid as a substrate. The surface electrostatic potential of the S. coelicolor PLA(2) model, which is created by the computer-homology modeling, suggests that the positively charged surface of the enzyme does not affect the substrate specificity.
Jovel
Santa Romero
SR
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Kumagai
Takanori
T
Danshiitsoodol
Narandalai
N
Matoba
Yasuyuki
Y
Nishimura
Motohiro
M
Sugiyama
Masanori
M
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D054467:Phospholipases A2; D017510:Protein Folding; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D048372:Streptomyces coelicolor / Q000201:enzymology*
D010741:Phospholipases A; D054467:Phospholipases A2