Primary structure, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus

The complete primary structure of inorganic pyrophosphatase [EC 3.6.1.1] fr om Bacillus stearothermophilus (ATCC 12016) was determined at the amino aci d level by automated Edman degradation. The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18,796. The amino acid sequence of the enzyme is almost identical to that of thermophil ic bacterium PS-3, Based on the determined primary structure, a PCR-amplifi ed semi-synthetic gene was constructed and expressed in Escherichia coli JM 109. The recombinant Bst. PPase showed the same characteristics and activit y as the authentic enzyme, and exhibits higher thermostability than the E. coli enzyme. Furthermore, we prepared tyrosine-substituted variants by site -directed mutagenesis to elucidate the role of two highly conserved tyrosin es (Y46 and Y130). As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected. However , the wild-type and two variants exhibited different thermostability behavi ors in the presence or absence of Mg2+. Therefore, these tyrosines may cont ribute to the structural integrity of the active site of the enzyme.