Genômica e biologia de fungos
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Abstrato

Characterization of Glucokinase-1 from Phytophthora infestans: A Versatile Enzyme

Liara Villalobos-Pina, Hermes Balza, Hector Acosta, Diego Andrade-Alviarez, Ascanio Rojas, Luisana Avilan, Amaranta Gomez-Arreaza

Phytophthora infestans has seven genes coding for glucokinases, with the [PITG_06016] (PiGlcK-1) gene being the highest expression at different stages of growth. However, its role in the metabolism of P. infestans is still unclear. In this study we cloned, overexpressed, purified and biochemically characterized this enzyme. The purified recombinant protein has a Km of 0.66 mM and 2.2 mM for glucose and ATP, respectively. PiGlcK-1 is capable of phosphorylating fructose, although only 48% with respect to glucose, using ADP and pyrophosphate (PPi) as a phosphoryl group donor. These molecules are 50% and 11% less effective than ATP, respectively. In addition, PiGlcK-1 can be activated by PPi increasing its activity 3.6 or 2.5 times when the phosphoryl donor is ATP or ADP, respectively. PiGlcK-1 can form oligomeric complexes and these tend to spontaneously dissociate, with the monomer having the highest activity. This oligomerization can be destabilized by dithiothreitol which increase PiGlcK-1 activity twice. These results reveal that PiGlcK-1 is a versatile enzyme that could play an important role in the use of available sugars during the first stage of infection, as well as in the necrotrophic stage, and suggest a possible regulatory mechanism against possible conditions of oxidative stress.