1887

Abstract

The genome of the thermophilic green-sulfur bacterium TLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (, 993 bp) and CT1713 (, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures – the largest one has an extra C-terminal HD domain – which may represent ancient paralogues. Both genes were cloned and overexpressed in BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg. Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P (polyP mix with mean chain length of 13–18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world.

Funding
This study was supported by the:
  • Spanish Government (Award BFU2004-00843, BFU2007-61887 and BFU2010-15622)
  • Andalusian Regional Government
  • EU FEDER
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2014-09-01
2021-10-18
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