Browsing by Author "Couto, Ana"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
- Bifunctional Homodimeric Triokinase/FMN CyclasePublication . Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José CarlosMammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.
- Reply to: Caution over the use of ecological big data for conservationPublication . Queiroz, Nuno; Humphries, Nicolas E.; Couto, Ana; Afonso, André S.; Sims, David W.; Afonso, Pedro; Bezerra, Natalia P. A.; Fontes, Jorge; Hazin, Fábio H.V.; Macena, Bruno C.L.; Travassos, Paulo; Vandeperre, Frederic; Vedor, Marisa; Costa, Iva do; Sequeira, Ana M.M.; Mucientes, Gonzalo; Santos, António M.; Abascal, Francisco J.; Abercrombie, Debra L.; Abrantes, Katya; Acuña-Marrero, David; Ferreira, Luciana C.; Ladino, Felipe; Lana, Fernanda O.; Quintero, Lina Maria; Duarte, Carlos M.
- Reply to: Shark mortality cannot be assessed by fishery overlap alonePublication . Queiroz, Nuno; Humphries, Nicholas E.; Couto, Ana; Afonso, André S.; Sims, David W.; Afonso, Pedro; Bezerra, Natalia P.A.; Fontes, Jorge; Hazin, Fábio H.V.; Macena, Bruno C.L.; Travassos, Paulo; Vandeperre, Frederic; Vedor, Marisa; Costa, Ivo da; Sequeira, Ana M.M.; Santos, António M.; Mucientes, Gonzalo; Abascal, Francisco J.; Abercrombie, Debra L.; Anders, Darrell; Araujo, Gonzalo