Abstract
Quaternary structure of CBS-pyrophosphatases (CBS-PPases), which belong to the PPases of family II, plays an important role in their function ensuring cooperative behavior of the enzymes. Despite an intensive research, high resolution structures of the full-length CBS-PPases are not yet available making it difficult to determine the signal transmission path from the regulatory to the active center. In the present work, small-angle X-ray scattering (SAXS) combined with size-exclusion chromatography was applied to determine the solution structures of the full-length wild-type CBS-PPases from three different bacterial species. Previously, in the absence of an experimentally determined full-length CBS-PPase structure, a homodimeric model of the enzyme based on known crystal structures of the CBS domain and family II PPase without this domain has been proposed. Our SAXS analyses demonstrate, for the first time, the existence of stable tetramers in solution for all studied CBS-PPases from different sources. Our findings show that further studies are required to establish the functional properties of these enzymes. This is important not only to enhance our understanding of the relation between CBS-PPases structure and function under normal conditions but also because some human pathogens harbor this class of enzymes.
Highlights
Inorganic pyrophosphatases (PPases) are enzymes present in all living organisms
The family II PPases are Co2+ or Mn2+ metalloenzymes that require Mg2+ for catalysis and the activities of CBS-PPases preincubated with 0.1 mM Co2+ are higher than those incubated with Mn2+
The catalytic site binding the nucleophile-coordinating metal cations is located at the N-terminal domain
Summary
Inorganic pyrophosphatases (PPases) are enzymes present in all living organisms. PPases catalyze hydrolysis of inorganic pyrophosphate into two molecules of orthophosphate, providing the necessary thermodynamic conditions for important reactions like protein-, RNA-, and DNA synthesis, and making these enzymes essential for life [1].Soluble PPases belong to three different non-homologous families: I, II, and III. Inorganic pyrophosphatases (PPases) are enzymes present in all living organisms. II PPases were discovered in 1998 [2,3] and are still intensively investigated. These enzymes exist almost exclusively in bacteria and archaebacteria, including human pathogens. Family II pyrophosphatases are composed of two well-defined domains connected by a flexible linker, N-terminal DHH and C-terminal DHHA2, and they belong to the DHH (Asp-His-His) family of phosphohydrolases [4]. The flexible linker between the N- and C-terminal domains and the active sites are located at the domain interfaces [5,6]. The C-terminal domain contains a high-affinity substrate-binding site, whereas the catalytic site that binds the nucleophile-coordinating metal cations is located at the N-terminal domain. Substrate binding to the C-terminal domain in its open conformation causes the domain closure [7]
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