Biochemical Characterization of Two Unrelated Microbial Proteins Involved on an in vitro Evolution System.

SANCHEZ, ALEJANDRO; CENTURIÓN, MARIO; FERREYRA, RAUL G.; ERMÁCORA, MARIO R.

San Miguel de Tucumán

Congreso; VII Congreso Argentino de Microbiología General SAMIGE 2011; 2011

Sociedad Argentina de Microbiología General SAMIGE

The amino acid sequence determines the three-dimensional structure of proteins. However, the relationship between sequence and structure is very complex and poorly understood. We have hypothesized that a sequence could in principle attain any structure, but the observed native conformation corresponds to the most energetically favorable structure. If this is true, any fold may be obtained from any sequence provided that additional appropriate mutations are introduced to stabilize the final desired structure. As a proof-of-concept, we set to obtain by in vitro evolution the chorismate mutase (CM) fold from the sequence of an unrelated hydrolase, the Bacillus licheniformis beta-lactamase (ESP). By genetic engineering, we placed six catalytic residues of B. subtillis CM into ESP and reduced the length of the latter from 265 to 157 residues. The resulting polypeptide (ESPCM) has the catalytic residues of CM properly spaced but in the context the ESP sequence. The conformational properties of ESPCM protein were characterized using biophysical and functional techniques. It is a molten globule devoid of tertiary structure and inactive for both, CM and lactamase, activities. Thus, ESPCM possesses all the prerequisites for the intended in vitro evolution. If ESPCM was to fold into the CM fold, it would have the catalytic residues of CM in the correct position in space and it would, in principle, possess CM activity. CM catalyzes the rearrangement of chorismate to yield prephenate, and this reaction is the first step of tyrosine and phenylalanine synthesis in bacteria and fungi. Exploiting this, we were able to complement a CM(-) Escherichia coli strain by transformation with an expression vector carrying the CM gene from the yeast Yarrowia lipolytica (YLCM), building a selection system that should allow us to detect and isolate in vitro evolved ESPCM mutants with CM activity. YLCM protein catalytic activity was also characterized by measuring its kinetics parameters. Along with the data presented, the implications of a positive result at the first round of evolution will be discussed.