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

Celina Frank,Dieter Jendrossek,Attila Teleki

doi:10.1007/s00253-020-10891-7

Celina Frank, Dieter Jendrossek + Show 1 more

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https://doi.org/10.1007/s00253-020-10891-7

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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, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT 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 PPK1AT is important for the formation of polyP whereas PPK2AT 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•PPK1ATand PPK2AThave different substrate specificities,•PPK2ATis a subgroup 1 member of PPK2s,•PPK2ATcatalyzes the formation of polyphosphorylated nucleosides

Highlights

Polyphosphate is an inorganic polymer in which phosphate residues are linked by energy-rich phosphoanhydride bonds
PPK1AT and PPK2AT eluted during nickel agarose affinity chromatography of soluble cell extracts at approximately 250 mM imidazole
Biochemical characterization of PPK1AT The substrate specificity of PPK1AT was analyzed by incubation of PPK1AT with ATP, GTP, CTP, dTTP, or UTP for 30 min and subsequent determination of the respective nucleotide compositions by HPLC

Summary

Introduction

Polyphosphate (polyP) is an inorganic polymer in which phosphate residues are linked by energy-rich phosphoanhydride bonds. It can be formed either abiotically (vulcanism) or biologically by the action of polyP kinases (PPKs in prokaryotes) or other enzymes (in eukaryotes). Two types of PPKs are known to catalyze the reversible formation of polyP from ATP: PPK1 type PPKs have a molecular mass of ≈ 80 kDa and consist of an N-terminal (N). PPKs of the PPK2 type have roughly only half of the molecular masses of PPK1s (MW around 40 kDa) and are divided into three subtypes dependent on their substrate specificities for nucleoside di-phosphates (subtype 1), nucleoside mono-phosphates (subtype 2), or both (subtype 3) (Motomura et al 2014). Several recent reports describe that some PPK2s are able to form nucleotides with four (Mordhorst et al 2019; Ogawa et al 2019; Hildenbrand et al 2019) or even five (Mordhorst et al 2019) phosphate residues

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