Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus.
Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-1, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49--54% identity, 76--81% homology). Taq FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40--50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent 21-amino-acid insertion sequence. The recombinant protein is thermostable for at least 45 min at 80 degrees C with little residual activity below 60 degrees C. Kinetic characterization at 70 degrees C, the optimal growth temperature for T. aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K(m) of 305 microM. The maximal specific activity (V(max)) is 46 U/mg at 70 degrees C.
Département de Biochimie, Université de Montréal, CP 6128, Succursale Centre Ville, Montréal, Québec, Canada H3C 3J7.
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase