Metabolite proofreading in carnosine and homocarnosine synthesis: molecular identification of PM20D2 as {beta}-alanyl-lysine dipeptidase [Metabolism]

June 2nd, 2014 by Veiga-da-Cunha, M., Chevalier, N., Stroobant, V., Vertommen, D., Van Schaftingen, E.

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-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 radiolabelled substrate, we found that rat skeletal muscle, heart and brain contained a cytosolic β-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 β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolysed β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine and γ-aminobutyryl-ornithine as its best substrates. It acted also, at lower rates, on β-alanyl-arginine and γ-aminobutyryl-arginine, but virtually not on carnosine or homocarnosine. Though acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted, at lower rates on some ′classical dipeptides′ like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was about 100 to 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 (β-alanyl-lysine, β-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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