Rat liver serine dehydratase. Bacterial expression and two folding domains as revealed by limited proteolysis.
A pCW vector harboring rat liver serine dehydratase cDNA was expressed in Escherichia coli. The expressed level was about 5-fold higher in E. coli BL21 than in JM109 cell extract; the former lacked two kinds of proteases. Immunoblot analysis revealed the occurrence of a derivative other than serine dehydratase in the JM109 cell extract. The recombinant enzyme was purified to homogeneity. Staphylococcus aureus V8 protease and trypsin cleaved the enzyme at Glu-206 and Lys-220, respectively, with a concomitant loss of enzyme activity. Spectrophotometrically, the nicked enzyme showed a approximately 50% reduced capacity for binding of the coenzyme pyridoxal phosphate and no spectral change of circular dichroism in the region at 300-480 nm, whereas circular dichroism spectra of both enzymes in the far-UV region were similar, suggesting that proteolysis impairs the coenzyme binding without an accompanying gross change of the secondary structure. Whereas the nicked enzyme behaved like the intact enzyme on Sephadex G-75 column chromatography, it was dissociated into two fragments on the column containing 6 M urea. Upon the removal of urea, both fragments spontaneously refolded. These results suggest that serine dehydratase consists of two folding domains connected by a region that is very susceptible to proteases.
Ogawa
H
H
Department of Biochemistry, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan. hogawa@ms.toyama-mpu.ac.jp
Takusagawa
F
F
Wakaki
K
K
Kishi
H
H
Eskandarian
M R
MR
Kobayashi
M
M
Date
T
T
Huh
N H
NH
Pitot
H C
HC
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D012695:L-Serine Dehydratase / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011732:Pyridoxal Phosphate / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D012695:L-Serine Dehydratase