Identification of a distinct family of polyphosphate kinases relevant to energy homeostasis in Crenarchaeota

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, occurs in all three domains of life and plays key roles in metabolism and regulation. While polyP metabolism has been well studied in bacteria and eukaryotes, studies in archaea have been limited, where the polyphosphate kinases (PPKs) involved in polyP synthesis remained largely uncharacterized. Notably, members of the Crenarchaeota (Thermoproteota) lack homologs of bacterial PPKs. We identify two genes in the crenarchaeal model organism Sulfolobus acidocaldarius, (saci_2019 and saci_2020), previously annotated as thymidylate kinases, that together encode a heteromeric archaeal PPK (SaPPK3). Saci_2019 acts as the catalytic subunit (cPPK3), whereas Saci_2020 is a regulatory subunit (rPPK3) that enhances activity through oligomerization. SaPPK3 is reversible but strongly favors polyP-dependent nucleotide kinase activity, forming ATP from ADP and polyP. Kinetic modelling combined with quantitative ³¹P NMR showed that polyP synthesis occurred only at high ATP/ADP ratios in the presence of an ATP recycling system, suggesting that SaPPK3 promotes ATP production from polyP under low energy conditions in vivo. Enzymatic, structural and phylogenetic analyses place SaPPK3 in a distinct PPK family within the thymidylate kinase superfamily of P-loop kinases. The PPK3 family members show a patchy distribution, being represented mainly in Crenarchaeota of the families Nitrososphaeraceae and Sulfolobaceae, and in a few bacteria. Our findings identify PPK3 as a critical missing link enzyme involved in archaeal polyP metabolism and suggests that polyP, in addition to its function in phosphate storage, might serve as an emergency energy buffer.