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

• Published in: Microbiology (Society for General Microbiology) 2014-12, Vol.160 (Pt 12), p.2807-2819
• Main Authors: Klemke, Friederike, Beyer, Gabriele, Sawade, Linda, Saitov, Ali, Korte, Thomas, Maldener, Iris, Lockau, Wolfgang, Nürnberg, Dennis J, Volkmer, Thomas
• Format: Article
• Language: English
• Subjects: Anabaena; Anabaena - enzymology; Anabaena - genetics; Anabaena - physiology; Cations, Divalent - metabolism; Cloning, Molecular; Cyanobacteria; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Deletion; Gene Expression; Gene Expression Profiling; Glucokinase - genetics; Glucokinase - metabolism; Glucose - metabolism; Hydrogen-Ion Concentration; Kinetics; Mannose - metabolism; Microbial Viability; Physiology and Biochemistry; Polyphosphates - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Temperature
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.081836-0

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.