Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.8/3027
Título: Bifunctional Homodimeric Triokinase/FMN Cyclase
Outros títulos: Contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements
Autor: Rodrigues, Joaquim Rui
Couto, Ana
Cabezas, Alicia
Pinto, Rosa María
Ribeiro, João Meireles
Canales, José
Costas, María Jesús
Cameselle, José Carlos
Palavras-chave: Animals
Catalysis
Catalytic Domain
Dimerization
Flavin-Adenine Dinucleotide
Fructose
Glyceraldehyde
Humans
Hydrogen-Ion Concentration
Kinetics
Molecular Dynamics Simulation
Mutagenesis, Site-Directed
Mutation
Phosphorus-Oxygen Lyases
Phosphorylation
Phosphotransferases (Alcohol Group Acceptor)
Protein Structure, Tertiary
Recombinant Proteins
Substrate Specificity
Data: 2014
Resumo: Mammalian 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.
Peer review: yes
URI: http://hdl.handle.net/10400.8/3027
DOI: 10.1074/jbc.M113.525626
ISSN: 1083-351X
Aparece nas colecções:Artigos em revistas internacionais

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J. Biol. Chem.-2014-Rodrigues-10620-36.pdf4,07 MBAdobe PDFVer/Abrir


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