Functional analysis of maize NADP-Malic enzyme chimeric proteins

ENRIQUE DETARSIO; MARIANA SAIGO; CLARISA ALVAREZ; CARLOS ANDREO; MARÍA FABIANA DRINCOVICH

Angra dos Reis

Congreso; 1st Latin American Protein Society Meeting; 2004

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), which catalyses the oxidative decarboxylation of L-malate to yield pyruvate, CO2 and NADPH,  is a widely distributed  enzyme involved in different metabolic pathways. At least two isoforms have been characterized in maize: photosynthetic (H) and non-photosynthetic (R ). The photosynthetic isoform is located in bundle sheath chloroplasts (BSC) in certain C4 plants such as maize, and provides CO2 to be fixed by RuBisCO. The non photosynthetic isoform is expressed in roots and leaves. The photosynthetic isoform seems to have evolved from the non-photosynthetic isoform, so the aim of this work is to associate the sequence differences with functional differences between them. These isoforms present two outstanding differences: first, only the photosynthetic form is inhibited at high concentrations of one of its substrates, L-malate. Second, although both exist in an equilibrium between a tetrameric and dimeric forms, the photosynthetic isoform is active as a tetramer but the non-photosynthetic as a dimer. In order to analyze the structural basis of these differences, chimeric proteins were constructed, with the following sequences: chimera HR 213 aa amino region photosynthetic isoform (H) + 289 aa carboxilic region of non photosynthetic isoform (R ), chimera RH: 216 aa amino region non-photosynthetic isoform (R) + 290 aa carboxilic region of photosynthetic isoform (H). The chimeric proteins were expressed in E.Coli, purified and showed very different specific activities: HR 92.8 UI/mg,  RH  0.24 UI/mg. Both chimeric proteins exhibited inhibition by L-malate, indicating that the this effect is not produced by the interaction of malate with a unique site present only in the photosynthetic isoform. On the other hand, the chimeric proteins showed low activity in their tetrameric forms. In conclusion, the lack of inhibition by malate and high activity in the tetrameric form requires aminoacids or structural features that are present in both halfs of the proteins R and H respectively.