The tetrameric structure of nucleotide-regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding

A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine β-synthase (CBS) domains and one DRTGG domain in addition to the two catalytic domains that form canonical Family II PPases. The CBS domain-containing PPases (CBS-PPases) are allosterically activated or inhibited by adenine nucleotides that cooperatively bind to the CBS domains. Here we use chemical cross-linking and analytical ultracentrifugation to show that CBS-PPases from Desulfitobacterium hafniense and four other bacterial species are active as 200–250-kDa homotetramers, which seems unprecedented among the four PPase families. The tetrameric structure is stabilized by Co2+, the essential cofactor, pyrophosphate, the substrate, and adenine nucleotides, including diadenosine tetraphosphate. The deletion variants of dhPPase containing only catalytic or regulatory domains are dimeric. Co2+ depletion by incubation with EDTA converts CBS-PPase into inactive tetrameric and dimeric forms. Dissociation of tetrameric CBS-PPase and its catalytic part by dilution renders them inactive. The structure of CBS-PPase tetramer was modelled from the structures of dimeric catalytic and regulatory parts. These findings signify the role of the unique oligomeric structure of CBS-PPase in its multifaced regulation. [Display omitted] •Adenine nucleotides regulate pyrophosphatases containing CBS domains (CBS-PPases).•Cross-linking and sedimentation data reveal homotetrameric structure of CBS-PPases.•Dissociation of CBS-PPase tetramer by dilution inactivates the enzyme.•Co2+ ions, substrate, and adenine nucleotides facilitate tetramerization of CBS-PPase.•Engineered separate catalytic and regulatory parts of CBS-PPase are dimeric.