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Metabolite proofreading in carnosine and homocarnosine synthesis: molecular identification of PM20D2 as ß-alanyl-lysine dipeptidase.
Veiga-da-Cunha, Maria; Chevalier, Nathalie; Stroobant, Vincent; Vertommen, Didier; Van Schaftingen, Emile.
Afiliação
  • Veiga-da-Cunha M; From the Welbio and the Laboratory of Physiological Chemistry, maria.veiga@uclouvain.be.
  • Chevalier N; From the Welbio and the Laboratory of Physiological Chemistry.
  • Stroobant V; the Ludwig Institute for Cancer Research, and.
  • Vertommen D; the Protein Phosphorylation Unit, de Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium.
  • Van Schaftingen E; From the Welbio and the Laboratory of Physiological Chemistry.
J Biol Chem ; 289(28): 19726-36, 2014 Jul 11.
Article em En | MEDLINE | ID: mdl-24891507
Carnosine synthase is the ATP-dependent ligase responsible for carnosine (ß-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic ß-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈ 200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with ß-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed ß-alanyl-lysine, ß-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on ß-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from ß-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ∼ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (ß-alanyl-lysine, ß-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Carnosina / Dipeptidases / Dipeptídeos Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2014 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Carnosina / Dipeptidases / Dipeptídeos Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2014 Tipo de documento: Article