Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus

Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermu s aquaticus YT-I, was cloned and sequenced. Nucleotide-sequence analysis re vealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple se quence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, A quifex aeolicus, and Helicobacter pylori (49-54% identity, 76-81% homology) . Tag FBP aldolase was overexpressed under tac promoter control in Escheric hia coli and purified to homogeneity using heat treatment followed by two c hromatographic steps. Yields of 40-50 mg of monodisperse protein were obtai ned per liter of culture. The quaternary structure is that of a homotetrame r stabilized by an apparent al-amino-acid insertion sequence, The recombina nt protein is thermostable for at least 45 min at 80 degreesC with little r esidual activity below 60 degreesC, Kinetic characterization at 70 degreesC , the optimal growth temperature for II aquaticus, indicates extreme negati ve subunit cooperativity (h = 0.32) with a limiting K-m of 305 muM. The max imal specific activity (V-max) is 46 U/mg at 70 degreesC. (C) 2001 Academic Press