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B6db references: 10212273

type Journal Article
authors Ogawa, H.; Takusagawa, F.; Wakaki, K., Kishi, H.; Eskandarian, M.R.; Kobayashi, M.; Date, T.; Huh, N.H.; Pitot H.C.
title Rat liver serine dehydratase. Bacterial expression and two folding domains as revealed by limited proteolysis
journal J Biol Chem
sel unselected
ui 10212273
year (1999)
volume 274
number 18
pages 12855-60.
abstract 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.
last changed 2004/06/14 15:57

B6db references