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Lookup NU author(s): Dr Zsolt Keresztessy, Emeritus Professor Monica Hughes
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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 (K-m) and the ratio between the maximal velocities and efficiency constants (V-max., V-max./K-m) 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(1)(E), pK(2)(E)) obtained with linamarin and PNP-Glc, and the enzyme substrate complexes (pK(1)(ES), pK(2)(ES)) 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 (k(i) = 0.007 min(-1)) and apparent inhibition constants (K-i = 20 mM) comparable viith 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.
Author(s): Keresztessy Z, Hughes J, Kiss L, Hughes MA
Publication type: Article
Publication status: Published
Journal: Biochemical Journal
Year: 1996
Volume: 314
Pages: 41-47
Print publication date: 01/02/1996
ISSN (print): 0264-6021
ISSN (electronic): 1470-8728
Publisher: Portland Press Ltd.
URL: http://www.biochemj.org/bj/314/bj3140041.htm
