Abstract
The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, with kcat = 48.2 s−1 at pH 7.5 and 28 °C and KM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.
Highlights
Inorganic polyphosphate, which is a linear polymer of 10– 1000 orthophosphates linked by phosphoanhydride bonds, has been found in all representative living cells, including bacteria, fungi, plants, animals and archaea (Achbergerova & Nahalka, 2011; Rao et al, 2009; Remonsellez et al, 2006; Scherer & Bochem, 1983)
Five of the identified domains have been proposed to interact with the ATP molecule: phosphate-1 and phosphate-2 are involved in binding b-phosphates and c-phosphates, while connect-1, connect-2 and the adenosine motif interact with the adenine ring of ATP (Mukai et al, 2003)
The glucose motif has been suggested to participate in glucose binding
Summary
Inorganic polyphosphate, which is a linear polymer of 10– 1000 orthophosphates linked by phosphoanhydride bonds, has been found in all representative living cells, including bacteria, fungi, plants, animals and archaea (Achbergerova & Nahalka, 2011; Rao et al, 2009; Remonsellez et al, 2006; Scherer & Bochem, 1983). Polyphosphate is stored in the cytoplasm, where it can be visualized as metachromic inclusions (Meyer, 1902) or electron-dense granules (Jensen, 1968). Polyphosphate stands as one of the earliest polymers produced in cells. Polyphosphate is considered to be the ancestor of ATP as an energy source (Resnick & Zehnder, 2000), given that hydrolysis of the Abbreviations: Anabaena, Anabaena sp. PCC 7120; GST, glutathione-Stransferase; PPGK, polyphosphate glucokinase; TSS, transcription start site. Two supplementary tables and five supplementary figures are available with the online Supplementary Material
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