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The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Å resolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Å resolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications. [ABSTRACT FROM AUTHOR]

Inorganic polyphosphate, a linear polymer of orthophosphate residues linked by phosphoanhydride bonds, occurs in all three domains of life and plays a diverse and prominent role in metabolism and cellular regulation. While the polyphosphate metabolism and its physiological significance have been well studied in bacteria and eukaryotes including human, there are only few studies in archaea available so far. In Crenarchaeota including members of Sulfolobaceae , the presence of polyphosphate and degradation via exopolyphosphatase has been reported and there is some evidence for a functional role in metal ion chelation, biofilm formation, adhesion and motility, however, the nature of the crenarchaeal polyphosphate kinase is still unknown. Here we used the crenarchaeal model organism Sulfolobus acidocaldarius to study the enzymes involved in polyphosphate synthesis. The two genes annotated as thymidylate kinase ( saci_2019 and saci_2020 ), localized downstream of the exopolyphosphatase, were identified as the missing polyphosphate kinase in S. acidocaldarius ( Sa PPK3). Thymidylate kinase activity was confirmed for Saci_0893. Notably Saci_2020 showed no polyphosphate kinase activity on its own but served as regulatory subunit (rPPK3) and was able to enhance polyphosphate kinase activity of the catalytically active subunit Saci_2019 (cPPK3). Heteromeric polyphosphate kinase activity is reversible and shows a clear preference for polyP-dependent nucleotide kinase activity, i.e. polyP-dependent formation of ATP from ADP (12.4 U/mg) and to a lower extent of GDP to GTP whereas AMP does not serve as substrate. PPK activity in the direction of ATP-dependent polyP synthesis is rather low (0.25 U/mg); GTP was not used as phosphoryl donor. A combined experimental modelling approach using quantitative 31 P NMR allowed to follow the reversible enzyme reaction for both ATP and polyP synthesis. PolyP synthesis was only observed when the ATP/ADP ratio was kept high, using an ATP recycling system. In absence of such a recycling system, all incubations with polyP and PPK would reach an equilibrium state with an ATP/ADP ratio between 3 and 4, independent of the initial conditions. Structural and sequence comparisons as well as phylogenetic analysis reveal that the S. acidocaldarius PPK is a member of a new PPK family, named PPK3, within the thymidylate kinase family of the P-loop kinase superfamily, clearly separated from PPK2. Our studies show that polyP, in addition to its function as phosphate storage, has a special importance for the energy homeostasis of S. acidocaldarius and due to its reversibility serves as energy buffer under low energy charge enabling a quick response to changes in cellular demand.