FUNCTIONAL ROLE OF GLU543 IN ALLOSTERIC FUMARATE REGULATION OF A. THALIANA NADP-MALIC ENZYME 2

”. ARIAS, CINTIA L.; GERRARD WHEELER, MARIEL C.; MAURINO, VERÓNICA G.; ANDREO, CARLOS S. AND DRINCOVICH, MARÍA F.

San Miguel de Tucumán

Congreso; XLV Reunión anual de la sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2009

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:Arial; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman"; color:black; mso-ansi-language:EN-US;} p {mso-margin-top-alt:auto; margin-right:0in; mso-margin-bottom-alt:auto; margin-left:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.35pt 842.0pt; margin:1.0in 283.5pt 1.0in 1.0in; mso-header-margin:1.0in; mso-footer-margin:1.0in; mso-paper-source:0;} div.Section1 {page:Section1;} --> A. thaliana contains four NADP-malic enzymes (ME 1-4) that share a high degree of sequence similarity but differ in tissue and cellular compartment expression, kinetic properties and metabolic regulation. ME2 is the most regulated isoform and we found out that Arg115 is necessary for its activation by fumarate. However, this residue is conserved in others malic enzymes, activated or not by this compound. Recent studies with chimerical proteins determined that the region from aminoacid 303 to C-terminal end of ME2 is responsible for fumarato activation in the forward reaction. Analysis of this sequence revealed that a Glu residue at position 543 is always present in activated isoforms, but not in inhibited or not regulated isoenzymes. Therefore, mutated versions of ME2, named E543K and E543A, were generated by site-direct mutagenesis. The two mutants were expressed in E. coli, purified and analysed. The regulation by fumarato of both mutants was affected by the substitution, however the effect was more pronounced in ME2E543K in which Glu543 was replaced for Lys changing the charge of the site. In this way, our results allowed the characterization of a new residue involved in the activation by fumarate in ME2. This regulation would be important in vivo in Arabidopsis, which accumulate great amounts of fumarate, to levels comparable to those of starch and soluble sugars.