Formation of an Organic–Inorganic Biopolymer: Polyhydroxybutyrate–Polyphosphate

Abstract

A considerable variety of different biopolymers is formed by the entirety of organisms present on earth. Most of these compounds are organic polymers such as polysaccharides, polyamino acids, polynucleotides, polyisoprenes or polyhydroxyalkanoates (PHAs), but some biopolymers can consist of solely inorganic monomers such as phosphate in polyphosphates (polyPs). In this contribution, we describe the formation of an organic–inorganic block copolymer consisting of poly­(3-hydroxybutyrate) (PHB) and polyP. This was achieved by the expression of a fusion of the polyP kinase gene (ppk2c) with the PHB synthase gene (phaC) of Ralstonia eutropha in a polyP-free and PHB-free mutant background of R. eutropha. The fusion protein catalyzed both the formation of polyP by its polyP kinase domain and the formation of PHB by its PHB synthase domain. It was also possible to synthesize the polyP-PHB polymer in vitro with purified Ppk2c-PhaC, if the monomers, adenosine triphosphate (ATP) and 3-hydroxybutyryl-CoA (3HB-CoA), were provided. Most likely, the formed block copolymer (polyP-protein-PHB) turns into a blend of polyP and PHB after release from the enzyme.

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

“…Purifications of PPK1 AT with a C-terminal and PPK2 AT with an N-terminal hexa-histidine tag were done as previously described for PPK2c of Ralstonia eutropha (Hildenbrand et al 2019). Purified PPK proteins (1-4 mg/ml) were stored on ice directly in elution buffer (pH 8.0 (composition see below) without any further treatment (250 mM imidazole, 300 mM NaCl and 50 mM NaH 2 PO 4 ).…”

“…Bacteria can have only the PPK1 type of PPKs, only the PPK2 type of PPKs, or both types of PPKs (PPK1 and PPK2) (Rao et al 2009). 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.…”

“…Indeed, two PPK1s (PPK1a and PPK1b) as well as five PPK2s (PPK2a-PPK2d) have been identified in R. eutropha (Tumlirsch et al 2015). One of the PPK2s, PPK2c, turned out to be highly active in vitro and catalyzed the formation of microscopically detectable polyP granules in vitro (Hildenbrand et al 2019;Hildenbrand et al 2020). The other PPKs of R. eutropha have not yet been investigated.…”

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate

“…The overexpression of ppk2c and purification of PPK2c with an N-terminal hexa-histidine tag was done as previously described (Hildenbrand et al 2019 ). Purified PPK2c (1.5 mg/ml) was shock-frozen in 0.2-ml aliquots in liquid nitrogen and stored at − 70 °C.…”

“…Indeed, two PPK1s (PPK1a and PPK1b) as well as five PPK2s (PPK2a – PPK2d) have been identified in R. eutropha (Tumlirsch et al 2015 ). One of the PPK2s, PPK2c, turned out to be highly active and catalysed the formation of microscopically detectable polyP granules in vitro, if ATP or GTP was present as a substrate (Hildenbrand et al 2019 ). In this study, we determined the biochemical properties of PPK2c and investigated the substrate specificity for different nucleoside-phosphates.…”