Cloning, expression, and characterization of the gsdA gene encoding thermophilic glucose-6-phosphate dehydrogenase from Aquifex aeolicus.
The gsdA gene of the extreme thermophilic bacterium Aquifex aeolicus, encoding glucose-6-phosphate dehydrogenase (G6PDH), was cloned into a high-expression vector and overexpressed as a fusion protein in Escherichia coli. Here we report the characterization of this recombinant thermostable G6PDH. G6PDH was purified to homogeneity by heat precipitation followed by immobilized metal affinity chromatography on a nickel-chelate column. The data obtained indicate that the enzyme is a homodimer with a subunit molecular weight of 55 kDa. G6PDH followed Michaelis-Menten kinetics with a K(M) of 63 micro M for glucose-6-phosphate at 70 degrees C with NADP as the cofactor. The enzyme exhibited dual coenzyme specificity, although it showed a preference in terms of k(cat)/ K(M) of 20.4-fold for NADP over NAD at 40 degrees C and 5.7-fold at 70 degrees C. The enzyme showed optimum catalytic activity at 90 degrees C. Modeling of the dimer interface suggested the presence of cysteine residues that may form disulfide bonds between the two subunits, thereby preserving the oligomeric integrity of the enzyme. Interestingly, addition of dithiothreitol or mercaptoethanol did not affect the activity of the enzyme. With a half-life of 24 h at 90 degrees C and 12 h at 100 degrees C, this is the most thermostable G6PDH described.
Iyer
Ramesh B
RB
Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
Wang
Jianquan
J
Bachas
Leonidas G
LG
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase