CHARACTERIZATION OF RECOMBINANT NADP DEPENDENT MALIC ENZYME FROM C3, C4 AND C3-C4 FLAVERIA SPECIES

SAAVEDRA DAMIAN DARÍO; DRINCOVICH MARÍA FABIANA; ANDREO CARLOS SANTIAGO

Mar del Plata

Congreso; XLIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

SAIB XLIII Reunión Annual Mar del Plata 2007PL-CO 18CHARACTERIZATION OF RECOMBINANT NADP DEPENDENT MALIC ENZYME FROM C3, C4 AND C3-C4 FLAVERIA SPECIESSaavedra, D.D.; Drincovich, M.F.; Andreo, C.S. CEFOBI. Facultad de Cs Bioquímicas y Farmacéuticas. UNR E-mail: saavedra@cefobi.gov.ar NADP-malic enzyme (NADP-ME) is a widely distributed enzyme involved in different metabolic pathways. The photosynthetic isoforms of this enzyme has evolved from non-photosynthetic counterparts. In order to analyze the origin of the C4-NADP-ME specific isoform, cDNAs encoding this enzyme were isolated in the genus Flaveria. This genus is well suited for studying the evolution of photosynthesis because it contains a continuous range of species between C3 and C4. In the present work, four cDNAs corresponding to plastidic isoforms from C3-C4 F. floridana, C4?like F. palmeri and C4 F. bidentis and F. trinervia species were isolated by RACE method and sequenced. Although these sequences share a high degree of identity, the phylogenetic tree constructed with plant NADP-MEs show that Flaveria isoforms group into two separate branches, corresponding to photosynthetic and non-photosynthetic isoforms. The complete codifying sequences of Flaveria NADP-ME were subcloned into the pET 32 expression vector and successfully expressed in E. coli. Recombinant proteins were purified and kinetically and structurally characterized. The results obtained indicate that, in spite of the high degree of similarity, these proteins display differences in optimum pH, km values and regulation, which may be important for their specific physiological function in vivo and related to the evolution towards C4-NADP-ME.