title: Unique PFK regulatory property from some mosquito vectors of disease, and from Drosophila melanogaster.
abstract: BACKGROUND
  Arthropod-borne diseases are some of the most rapidly spreading diseases. Reducing the vector population is currently the only effective way to reduce case numbers. Central metabolic pathways are potential targets to control vector populations, but have not been well explored to this aim. The information available on energy metabolism, as a way to control lifespan and dispersion through flight of dipteran vectors, is inadequate.
  
  
  METHODS
  Phosphofructokinase (PFK) activity was measured in the presence of both of its substrates, fructose-6-phosphate (F6P) and ATP, as well as some allosteric effectors: Fructose- 2,6 - bisphosphate (F2, 6BP), citrate and AMP. Aedes aegypti phosphofructokinase sequence (AaPFK) was aligned with many other insects and also vertebrate sequences. A 3D AaPFK model was produced and docking experiments were performed with AMP and citrate.
  
  
  RESULTS
  The kinetic parameters of AaPFK were determined for both substrates: F6P (V = 4.47 ± 0.15 μmol of F1, 6BP/min, K0.5 = 1.48 ± 0.22 mM) and ATP (V = 4.73 ± 0.57 μmol of F1, 6BP/min, K0.5 = 0.43 ± 0.10 mM). F2,6P was a powerful activator of AaPFK, even at low ATP concentrations. AaPFK inhibition by ATP was not enhanced by citrate, consistent with observations in other insects. After examining the sequence alignment of insect and non-insect PFKs, the hypothesis is that a modification of the citrate binding site is responsible for this unique behavior. AMP, a well-known positive effector of PFK, was not capable of reverting ATP inhibition. Aedes, Anopheles and Culex are dengue, malaria and filariasis vectors, respectively, and are shown to have this distinct characteristic in phosphofructokinase control. The alignment of several insect PFKs suggested a difference in the AMP binding site and a significant change in local charges, which introduces a highly negative charge in this part of the protein, making the binding of AMP unlikely. This hypothesis was supported by 3D modeling of PFK with AMP docking, which suggested that the AMP molecule binds in a reverse orientation due to the electrostatic environment. The present findings imply a potential new way to control PFK activity and are a unique feature of these Diptera.
  
  
  CONCLUSIONS
  The present findings provide the first molecular explanation for citrate insensitivity in insect PFKs, as well as demonstrating for the first time AMP insensitivity in dipterans. It also identified a potential target for novel insecticides for the control of arthropod-borne diseases.
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. rdnunes@bioqmed.ufrj.br.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil. rdnunes@bioqmed.ufrj.br.
authors.forename: Rodrigo Dutra
authors.initials: RD
authors.lastname: Nunes
authors.affiliation: Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Nelilma@iq.ufrj.br.|NUPEM-Macaé, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Nelilma@iq.ufrj.br.
authors.forename: Nelilma Correia
authors.initials: NC
authors.lastname: Romeiro
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Riode Janeiro, RJ, Brazil. hugo.carvalho@ime.uerj.br.
authors.forename: Hugo Tremonte
authors.initials: HT
authors.lastname: De Carvalho
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Riode Janeiro, RJ, Brazil. je.ri@hotmail.com.
authors.forename: Jean Ribeiro
authors.initials: JR
authors.lastname: Moreira
authors.affiliation: Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. maurosp@pharma.ufrj.br.
authors.forename: Mauro
authors.initials: M
authors.lastname: Sola-Penna
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. maneto@bioqmed.ufrj.br.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil. maneto@bioqmed.ufrj.br.
authors.forename: Mário Alberto C
authors.initials: MA
authors.lastname: Silva-Neto
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Riode Janeiro, RJ, Brazil. gbraz@iq.ufrj.br.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil. gbraz@iq.ufrj.br.
authors.forename: Glória Regina Cardoso
authors.initials: GR
authors.lastname: Braz
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002951:Citrates / Q000378:metabolism; D009033:Culicidae / Q000201:enzymology* / Q000502:physiology*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000502:physiology*; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D007303:Insect Vectors; D007700:Kinetics; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D002951:Citrates; D005635:Fructosediphosphates; D005636:Fructosephosphates; D000249:Adenosine Monophosphate; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1