In order to identify and assess sequence markers that support structure and specificity, we have undertaken the study of a large enzyme superfamily, comprised mostly of phosphotransferases, the haloalkanoate dehalogenase superfamily (HADSF). Because of the occurrence of the family in all domains of life and the number of homologues within each organism the members provide numerous examples of orthologues to study determinants of specificity and paralogues to study function diversification. The HADSF has successfully evolved several forms of chemical transformation and has experienced expansion through substrate space. The expansion includes the invention and reinvention (pseudoconvergent evolution) of activities within branches of the family. Notably, members show substrate ambiguity, with activity toward a number of substrates and significant substrate overlap between “paralogues”. Other family members have been honed to a specific substrate with high catalytic efficiency and proficiency. However, substrate ambiguity can also be associated with high catalytic efficiency. In order to obtain a “panoramic view” of the superfamily, we have employed high throughput substrate screening to a diverse set of HADSF enzymes from a sampling of bacteria across phyla. The results highlight that substrate ambiguity is much more prevalent than one might imagine with ~45% of all proteins showing activity against multiple substrates. The structural basis of promiscuity in the HADSF highlights the necessity of a binding surface with multiple enzyme candidate residues to provide potential ligands, provided in the HADSF by the insertion of a cap domain into the Rossmann core fold. Overall, our findings are consistent with the concept that domain insertions act to increase the substrate range of the superfamily.

Adaptation of phosphatases as regulators, catalysts, and housekeepers

254th National Meeting (2017)

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