A pseudo-beta-glucosidase in Arabidopsis thaliana: correction by site-directed mutagenesis, heterologous expression, purification, and characterization.
Since At2g25630 is an intronless gene with a premature stop codon, its cDNA encoding the predicted mature beta-glucosidase isoenzyme was synthesized from the previously isolated Arabidopsis thaliana genomic DNA. The stop codon was converted to a sense codon by site-directed mutagenesis. The native and mutated cDNA sequences were separately cloned into the vector pPICZalphaB and expressed in Pichia pastoris. Only the cells transformed with mutated cDNA-vector construct produced the active protein. The mutated recombinant beta-glucosidase isoenzyme was chromatographically purified to apparent homogeneity. The molecular mass of the protein is estimated as ca. 60 kD by SDS-PAGE. The pH optimum of activity is 5.6, and it is fairly stable in the pH range of 5.0-8.5. The purified recombinant beta-glucosidase is effectively active on para-/ortho-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with K(m) values of 1.9, 2.1, 0.78 mM and k(cat) values of 114, 106, 327 nkat/mg, respectively. It also exhibits different levels of activity against para-/ortho-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), amygdalin, prunasin, cellobiose, gentiobiose, and salicin. The enzyme is competitively inhibited by gluconolactone and p-nitrophenyl-1-thio-beta-D-glucopyranoside with p-NPG, o-NPG, and 4-MUG as substrates. The enzyme is found to be very tolerant to glucose inhibition. The catalytic role of nucleophilic glutamic acid in the motif YITENG of beta-glucosidases and mutated recombinant enzyme is discussed.
Turan
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Balikesir University, Arts and Sciences Faculty, Department of Biology, Cagis Kampusu, Balikesir, Turkey. yturan3@yahoo.com
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D001617:beta-Glucosidase