Structure and Function of BorB, the Type II Thioesterase from the Borrelidin Biosynthetic Gene Cluster

α/β hydrolases make up a large and diverse protein superfamily. In natural product biosynthesis, <i>cis</i>-acting thioesterase α/β hydrolases can terminate biosynthetic assembly lines and release products by hydrolyzing or cyclizing the biosynthetic intermediate. Thioesterases can also act in <i>trans</i>, removing aberrant intermediates and restarting stalled biosynthesis. Knockout of this "editing" function leads to reduced product titers. The borrelidin biosynthetic gene cluster from <i>Streptomyces parvulus</i> Tü4055 contains a hitherto uncharacterized stand-alone thioesterase, <i>borB</i>. In this work, we demonstrate that purified BorB cleaves acyl substrates with a preference for propionate, which supports the hypothesis that it is also an editing thioesterase. The crystal structure of BorB shows a wedgelike hydrophobic substrate binding crevice that limits substrate length. To investigate the structure-function relationship, we made chimeric BorB variants using loop regions from characterized homologues with different specificities. BorB chimeras slightly reduced activity, arguing that the modified region is a not major determinant of substrate preference. The structure-function relationships described here contribute to the process of elimination for understanding thioesterase specificity and, ultimately, engineering and applying <i>trans</i>-acting thioesterases in biosynthetic assembly lines.