Biophysical and Biochemical Characterization of Chorismate Mutase from the Yeast Yarrowia lipolytica

SIERRA, IVANA; SANCHEZ, ALEJANDRO; FERREYRA, RAUL G.; ERMÁCORA, MARIO R.

San Javier, Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica SAB

Chorismate mutase (CM), a branch-point enzyme in the aromatic amino acid pathway, catalyzes the rearrangement of chorismate to yield prephenate, and this reaction is the first step of tyrosine and phenylalanine synthesis in plant, bacteria and fungi .Understanding how CM works can be useful for subsequent designing of modifications that allow phenylalanine and tyrosine overproduction. The fact that CM is found only in fungi, bacteria and higher plants makes it a target for the creation of antimicrobial as well as herbicides agents. In addition, the low sequence homology between known CM provides the opportunity to develop specific modulators of activity. Yarrowia lipolytica-CM (YLCM) is a 256 residues protein with 50 % homology to Saccharomyces cerevisiae CM. YLCM was expressed in Escherichia coli and by two different methods. The first included an anion exchange column (Q-Sepharose) equilibrated at basic pH and the other a cation exchange (SP-Sepharose) column at acidic pH. The conformational properties of purified YLCM were characterized using biophysical and functional techniques. YLCM protein catalytic activity was also characterized. The preliminary results demonstrate that the purification at basic pH provides a more stable YLCM, with larger native optical signals and specific activity than the protein from the acid purification.