Flores_2012_Protein.Eng.Des.Sel_25_387

Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual involving simple amino acid residue substitutions or drastic such that larger structural elements are inserted deleted or rearranged. Vast protein sequence comparisons supported by some experimental evidence argue that large structural modifications have been necessary for certain catalytic activities to evolve. However it is not clear whether these activities could not have been attained by gradual changes. Interestingly catalytic promiscuity could play a fundamental evolutionary role a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzyme's primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift making a pool of potentially useful activities that can be selected before duplication In this work we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859 a small protein to attain the function of BioH a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.