Co-purification from Escherichia coli of a plant beta-glucosidase-glutathione S-transferase fusion protein and the bacterial chaperonin GroEL.
The coding sequence of the mature cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowski, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (London) 371, 578-586] was found to be tightly associated with the fusion protein and co-purified with it. In the presence of excess MgATP, release and folding of the fusion beta-glucosidase were demonstrated by a fast increase in both linamarase and p-nitrophenyl-beta-D-glucopyranosidase activity at a low protein concentration. A slow endogenous folding process was also detected by activity measurements. Michaelis constants (Km) and the ratio between the maximal velocities and efficiency constants (Vmax., Vmax./Km) for the hydrolysis of the natural substrate, linamarin, and p-nitrophenyl beta-D-glucopyranoside (PNP-Glc) by the recombinant protein were found to be almost identical with those of the native glycosylated plant enzyme [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 314, 142-152]. Molecular dissociation constants for the free enzyme (pK(E)1, pK(E)2) obtained with linamarin and PNP-Glc, and the enzyme substrate complexes (pK(ES)1, pK(ES)2) were also in accordance with that of the original protein. The reactive substrate analogue N-bromoacetyl beta-D-glucosylamine inactivated the fusion enzyme according to pseudo-first-order kinetics with first-order rate constant (k1=0.007 min-1) and apparent inhibition constants (k1=20 mM) comparable with those of the plant protein [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 315, 323-330]. In comparison with the native glycosylated plant protein, the recombinant protein was, however, found to be extremely sensitive to proteolysis and misfolding.
Keresztessy
Z
Z
Department of Biochemistry and Genetics, University of Newcastle upon Tyne, U.K.
Hughes
J
J
Kiss
L
L
Hughes
M A
MA
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D002365:Manihot / Q000201:enzymology*; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018834:Chaperonin 60; D017931:DNA Primers; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; C090780:N-bromoacetylglucopyranosylamine; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; C021514:cyanogenic beta-glucosidase; D001617:beta-Glucosidase; D000117:Acetylglucosamine