CLONING AND CHARACTERIZATION OF NADP DEPENDENT MALIC ENZYME FROM C3 AND C4 FLAVERIA SPECIES

SAAVEDRA DAMIAN DARÍO; DRINCOVICH MARÍA FABIANA; ANDREO CARLOS SANTIAGO

Posadas

Congreso; XL Reunión Anual SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR; 2004

CLONING AND CHARACTERIZATION OF NADP DEPENDENT MALIC ENZYME FROM C3 AND C4 FLAVERIA SPECIESSaavedra, Damián D.; Drincovich, María F. and Andreo, Carlos S. CEFOBI. Facultad de Cs Bioquímicas y Farmacéuticas. UNR. Suipacha 531. 2000. Rosario. Argentina. e-mail: dsaavedr@fbioyf.unr.edu.arC4 plants have evolved independently from C3 ancestral species many times during the evolution of angiosperms. NADP-malic enzyme (NADP-ME) is a widely distributed enzyme involved in different metabolic pathways. The photosynthetic isoform of this enzyme is found in the bundle sheath chloroplasts of certain C4 plants and has evolved from non-photosynthetic isoforms. In order to analyze the origin of the C4-specific isoform of NADP-ME within the dicots, we isolated cDNAs encoding this enzyme in the genus Flaveria. This genus is well suited for studying the evolution of photosynthesis because it contains a more or less continuous range of species between C3 and C4, including C3-C4 and C4-like species. In the present work, a suitable method to isolate RNA from Flaveria tissue was developed. Then, the full length cDNAs of ChlME1 (codifying a NADP-ME plastidic isoform from the C4 F. bidentis) and modA (codifying a NADP-ME plastidic isoform from the C3 F. pringlei) were isolated by RT-PCR. The amplified products were cloned into pGEM-T, sequenced and subcloned into the pET 32 expression vector. The recombinant proteins were expressed in E. coli, purified and used for kinetic and structural characterizations. The results obtained indicate that, although both genes show a high degree of homology, the proteins display several kinetic and structural differences, which may be important for their specific physiological function in vivo.