Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates

ABSTRACT: Agrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1(AT) and ppk2(AT), of which only ppk1(AT) is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1(AT) and PPK2(AT) proteins revealed a higher substrate specificity of PPK1(AT) (in particular for adenine nucleotides) than for PPK2(AT). In contrast, PPK2(AT) accepted all nucleotides at comparable rates. Most interestingly, PPK2(AT) catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data—in combination with in vivo results—suggest that PPK1(AT) is important for the formation of polyP whereas PPK2(AT) has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification. KEY POINTS: •PPK1(AT) and PPK2(AT) have different substrate specificities, •PPK2(AT) is a subgroup 1 member of PPK2s, •PPK2(AT) catalyzes the formation of polyphosphorylated nucleosides ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00253-020-10891-7) contains supplementary material, which is available to authorized users.