Two strictly polyphosphate-dependent gluco(manno)kinases from diazotrophic Cyanobacteria with potential to phosphorylate hexoses from polyphosphates.
The single-copy genes encoding putative polyphosphate-glucose phosphotransferases (PPGK, EC 2.7.1.63) from two nitrogen-fixing Cyanobacteria, Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102, were cloned and functionally characterized. In contrast to their actinobacterial counterparts, the cyanobacterial PPGKs have shown the ability to phosphorylate glucose using strictly inorganic polyphosphates (polyP) as phosphoryl donors. This has proven to be an economically attractive reagent in contrast to the more costly ATP. Cyanobacterial PPGKs had a higher affinity for medium-long-sized polyP (greater than ten phosphoryl residues). Thus, longer polyP resulted in higher catalytic efficiency. Also in contrast to most their homologs in Actinobacteria, both cyanobacterial PPGKs exhibited a modest but significant polyP-mannokinase activity as well. Specific activities were in the range of 180-230 and 2-3 μmol min(-1) mg(-1) with glucose and mannose as substrates, respectively. No polyP-fructokinase activity was detected. Cyanobacterial PPGKs required a divalent metal cofactor and exhibited alkaline pH optima (approx. 9.0) and a remarkable thermostability (optimum temperature, 45 °C). The preference for Mg(2+) was noted with an affinity constant of 1.3 mM. Both recombinant PPGKs are homodimers with a subunit molecular mass of ca. 27 kDa. Based on database searches and experimental data from Southern blots and activity assays, closely related PPGK homologs appear to be widespread among unicellular and filamentous mostly nitrogen-fixing Cyanobacteria. Overall, these findings indicate that polyP may be metabolized in these photosynthetic prokaryotes to yield glucose (or mannose) 6-phosphate. They also provide evidence for a novel group-specific subfamily of strictly polyP-dependent gluco(manno)kinases with ancestral features and high biotechnological potential, capable of efficiently using polyP as an alternative and cheap source of energy-rich phosphate instead of costly ATP. Finally, these results could shed new light on the evolutionary origin of sugar kinases.
Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, CSIC y Universidad de Sevilla, Av. Américo Vespucio 49, 41092, Sevilla, Spain.
Tomás
T
Albi
Aurelio
A
Serrano
D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006601:Hexoses / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D046937:Nostoc / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011122:Polyphosphates / Q000378:metabolism*; D055503:Protein Multimerization; D013696:Temperature
D003067:Coenzymes; D006601:Hexoses; D011122:Polyphosphates; D017853:Phosphotransferases (Alcohol Group Acceptor); D005941:Glucokinase; C032922:mannokinase