All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120

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.

Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels

“…2A). We took advantage of the existence of strictly polyP-dependent glucokinase (PPGK) enzymes from cyanobacteria that cannot use nucleoside triphosphates (NTPs) as phosphodonors (16,17). We codon optimized the gene for one such enzyme, all1371 (here referred to as ppgK), from Anabaena sp.…”

“…We codon optimized the gene for one such enzyme, all1371 (here referred to as ppgK), from Anabaena sp. strain PCC 7120 (17), for expression in E. coli and cloned it into a plasmid with an arabinose-inducible promoter (72). E. coli has two glucose phosphorylation systems that catalyze the essential first step in assimilating glucose via glycolysis: the ATP-dependent cytoplasmic enzyme glucokinase (GLK; encoded by the glk gene) and the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which phosphorylates glucose during its transport across the cell membrane (73-75).…”

“…In vertebrates, in which the enzyme(s) of polyP synthesis is not yet known, polyP is involved in processes as diverse as blood clotting, inflammation, bone mineralization, neurotransmission, and amyloid protein aggregation (3)(4)(5)(6)(7). In bacteria, in which polyP is synthesized by the polyP kinase (PPK) (8,9), polyP has been shown to act as a protein-stabilizing chaperone, a regulator of protease activity, a chelator of heavy metals, an energy source, and a phosphate reservoir, and it plays important roles in a variety of stress responses, DNA damage repair, the cell cycle, motility, and biofilm formation (1,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Notably, loss of the PPK-encoding ppk gene causes a wide variety of bacterial pathogens to become avirulent (e.g., see references 22 to 28), so an improved understanding of bacterial polyP metabolism could have profound implications for the control of infectious diseases.…”

Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

“…PPGKs have been discovered in numerous organisms, such as mycobacterial species (11), Propionibacterium shermanii (12), Microlunatus phosphovorus (13), Arthrobacter spp. (14), Corynebacterium glutamicum (15), diazotrophic cyanobacterial species (16,17), and so on. However, nearly all reported PPGKs have been isolated from mesophilic sources, and the only thermophilic PPGK from Thermobifida fusca suffers from rapid deactivation at 50°C (i.e., half-life [t 1/2 ], 15 min) (2).…”

Intracellular accumulation of trehalose and glycogen in an extreme oligotroph, Rhodococcus erythropolis N9T-4

“…2). The polyP-dependent glucokinase found in several bacteria [14][15][16][17] may be involved in the synthesis of glucose 6-phosphate as one of the substrates for OtsA. The reason why the N9T-4 cells accumulate glycogen, trehalose, and polyP under these oligotrophic conditions is not clear.…”