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The N-terminal 15 amino acids of the major protein associated with inorganic pyrophosphatase activity in Bacillus subtilis WB600 are identical to those of B. subtilis ORF yybQ. This ORF was amplified from B. subtilis WB600 DNA by PCR and cloned into an overexpression vector in Escherichia coli. Induction of overexpression produced a soluble protein of 34000 Da by SDS-PAGE and by matrix-assisted laser desorption and ionization mass spectrometry. The overexpressed protein had a high specific activity for the hydrolysis of magnesium pyrophosphate, and was specifically and reversibly activated by Mn2+ ions. These properties are identical to those of inorganic pyrophosphatase purified from B. subtilis WB600. No significant similarity was found between the derived sequence of the B. subtilis yybQ-encoded protein and published sequences of identified inorganic pyrophosphatases of Eukarya, Bacteria or Archaea domains. However, there is significant similarity to three putative proteins of unknown function from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from Streptococcus gordonii. The genomes of B. subtilis, M. jannaschii and A. fulgidus do not contain sequences similar to those of hitherto known soluble inorganic pyrophosphatases. The present findings, together with a survey of the properties of inorganic pyrophosphatases from 38 different sources, suggest that the B. subtilis yybQencoded protein is the first fully characterized member of a new class of inorganic pyrop hosp hatase.I