Directed evolution of a beta-lactamase with a shorter Omega-loop

In protein engineering and directed evolution of enzymes, many publications report changes in substrate specificity, but rare are the birth of new enzyme activity. Through point mutations and gradual changes in substrate specificity, many evolved proteins have been described. Nevertheless, few studies address the question of the emergence of different catalytic activities within a superfamily. Beside protein evolution through single mutations, other mutagenic events are known to play crucial roles: duplication, insertions and deletions (InDels), domain shuffling, circular permutation, etc. However, researchers use mainly random point mutations for evolving proteins in the lab because efficient methods for generating random InDels and shuffling are lacking. InDels are observed when aligning protein superfamilies for which the tolerance of stable folds to variation in sequence and domain lengths is emphasised through the diversity of proteins sharing similar structures. The functional versatility is attributed to novel interfaces resulting from domain modulation of pre-existing scaffolds through length modifications introduced through insertions and deletions. In this regard, PBPs and class A beta- lactamases are both active serine enzymes superfamily with similarities regarding structural scaffolds and conserved active site motifs. Their catalytic activities are close in catalytic space, and all serine beta-lactamases are expected to have evolved from ancestral PBPs by acquiring a new catalytic element capable of activating a water molecule in the very crowded environment of the penicilloyl-enzyme. However, all attempts to emerge a beta-lactamase from a PBP were unsuccessful or gave very modest results. In this study, we investigate the essentiality and the evolutionary history of an essential omega-loop flanking the active site of class A beta-lactamases. Our results suggest that an ancestral insertion in this omega-loop probably occured for improving the enzyme efficiency but not as the key event in the beta-lactamase birth.