A Split Gene Approach to Alleviate Severe Inhibition of Catalysis by Substrate

Iodotyrosine deiodinases (IYDs) generally suffer from substrate inhibition and none more acutely than a homologue from <i>Thermotoga neapolitana</i> (TnIYD) that is an attractive target for engineering a robust catalysis to degrade halophenols that are persistent in the environment. The mechanism of this inhibition for TnIYD and the human homologue is now shown to derive from the formation of a nonproductive complex between the substrate 3-iodo-l-tyrosine and the enzyme in its oxidized form. This complex prevents subsequent reduction required for catalysis. Here, we describe a split gene approach to overcome this constraint. By coexpression, fragments of TnIYD assemble into an active catalyst with a discontinuity in the polypeptide backbone and deletion of residues from the active site lid. Three sets of enzymes were generated, and at least one representative of each overcomes substrate inhibition and demonstrates a gain in <i>k</i>_cat. Our results suggest that a significant fraction of the active site lid is dispensable and not necessary for promoting reductive dehalogenation. This approach offers a complement to circular permutation for manipulating the dynamics and accessibility of active sites.