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Arch Biochem Biophys. 1995 May 10;319(1):149-56.

Purification, cloning, and sequencing of archaebacterial pyrophosphatase from the extreme thermoacidophile Sulfolobus acidocaldarius.

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Institute of Biochemistry, Medical University of Lübeck, Germany.


Cytoplasmic pyrophosphatases are indispensible for the function of cellular bioenergetics. From the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius, situated at one of the lowest branches of the phylogenetic tree, a cytosolic pyrophosphatase has been isolated and purified 200-fold to electrophoretic homogeneity by combining ion-exchange and gel-exclusion chromatography. The native enzyme consists of a homotetramer of 71 kDa apparent molecular mass; the subunit displays an apparent molecular mass of 17 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an absolute requirement for divalent cations (Mg2+) and a temperature optimum of 75 degrees C coinciding with the growth optimum of the organism; the apparent estimated activation energy is 79.5 kJ/mol. A large variety of cytosolic extracts from other archaebacteria has been probed with a polyclonal antiserum raised against the purified protein; surprisingly, except for an extremely weak signal with S. solfataricus none of the other organisms showed any cross-reactivity. Also, Escherichia coli PPase does not cross-react. Based on N-terminal sequencing the gene has been cloned and sequenced. It codes for a 173-amino-acid protein with a calculated molecular mass of 19,365 kDa. Alignment with known eucaryotic and procaryotic PPases reveals invariant conservation of all residues presently assumed to be involved in metal and substrate binding. Unexpectedly, the highest similarity is found with the enzyme from the phylogenetically extremely distant eubacterium E. coli, but immunological cross-reactivity is absent. Similarity to the only known other archaebacterial PPase is much weaker. Using the 3D structure of the Thermus thermophilus enzyme as a scaffold an energy-minimized structural model is presented, deviating only minimally from the former. The structural features are discussed. The enzyme provides an excellent model for studies of thermostability and folding dynamics since heterologous overexpression has been achieved and genetically mutated forms become accessible.

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