Bifunctional ADP-dependent phosphofructokinase/ glucokinase activity in the order Methanococcales – biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis
Author
dc.contributor.author
Castro Fernández, Víctor
Author
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Bravo Moraga, Felipe
es_CL
Author
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Herrera Morandé, Alejandra
es_CL
Author
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Guixé Leguía, Victoria Cristina
es_CL
Admission date
dc.date.accessioned
2014-12-12T18:09:00Z
Available date
dc.date.available
2014-12-12T18:09:00Z
Publication date
dc.date.issued
2014
Cita de ítem
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FEBS Journal 281 (2014) 2017–2029
en_US
Identifier
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DOI: 10.1111/febs.12757
Identifier
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https://repositorio.uchile.cl/handle/2250/119818
General note
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Artículo de publicación ISI
en_US
Abstract
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In some archaea, the phosphorylation of glucose and fructose 6-phosphate
(fructose 6P) is carried out by enzymes that are specific for either substrate
and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon
Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate
glucose and fructose 6P has been described. To determine whether the
ability to phosphorylate both glucose and fructose 6P is a common feature
for all enzymes of the order Methanococcales, we expressed, purified and
characterized the unique homologous protein of the mesophilic archaea
Methanococcus maripaludis. Assay of the enzyme activity with different
sugars, metals and nucleotides allows us to conclude that the enzyme is
able to phosphorylate both fructose 6P and glucose in the presence of
ADP and a divalent metal cation. Kinetic characterization of the enzyme
revealed complex regulation by the free Mg2+ concentration and AMP,
with the latter appearing to be a key metabolite. To determine whether this
enzyme could have a role in gluconeogenesis, we evaluated the reversibility
of both reactions and found that glucokinase activity is reversible, whereas
phosphofructokinase activity is not. To determine the important residues
for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/
glucokinase enzyme from M. maripaludis and its interactions
with both sugar substrates using protein–ligand docking. Comparison of
the active site of the phosphofructokinase/glucokinase enzyme from
M. maripaludis with the structural models constructed for all the homology
sequences present in the order Methanococcales shows that all of the ADPdependent
kinases from this order would be able to phosphorylate glucose
and fructose 6P, which rules out the current annotation of these enzymes
as specific phosphofructokinases.
en_US
Patrocinador
dc.description.sponsorship
This work was supported by Fondo Nacional de
Desarrollo Cientifico y Tecnologico (Fondecyt, Chile)
Grant 1110137 and CONICYT scholarship 24121448.
Bifunctional ADP-dependent phosphofructokinase/ glucokinase activity in the order Methanococcales – biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis