Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation, N-, and S-methylation

Abstract

The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity.

Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones

“…12,23 The results of the substrate proles showed that both Ecm6(AM s M b A) and Ecm6(AM b M s A) were very specic for L-Ser. This along with other engineering studies 12,13 indicate that the natural substrate selectivity is not dramatically altered by insertion of one or two M domains into the a8- it is known that A and T domains can be separated in NRPS systems and investigated independently, 24 some constructs are impaired by improper division of the A and T domains, 25,26 therefore we sought to verify that this was not the case with our engineered interrupted A domain constructs. We found that there was a negligible difference in the relative percent of the adenylation activity with L-Ser between the two enzyme sets (Fig.…”

“…This is a signicant challenge to overcome for engineering back-to-back interrupted A domains, which was not a problem when only one M domain was articially inserted. 12,13 Until there is a crystal structure solved, we will not know for sure how close together the active sites are for back-to-back interrupted A domains. Alternatively, it could be that the two M domains used in this project were not compatible or did not communicate or coordinate well with each other or the A and T domains, halting methylation.…”

A thorough analysis and categorization of bacterial interrupted adenylation domains, including previously unidentified families

“…We could envision that an interrupted A domain of this nature, based on the trends we have observed here, would contain an M s( S ,a2–a3) , as naturally found paired with an M b(a8–a9) interruption. This di-interruption is functionally possible as we showed this conformation can exist artificially, 46 although we have yet to discover a natural di-interrupted A domain like this.…”

“…Previous work has shown the malleability of interrupted A domains, in that they can have their M domains exchanged, 47 be created from scratch, 48 and be created to possess unnatural domain arrangements (e.g., a2-a3 and a8-a9 di-interrupted A domains). 46 Most significantly, we have identified two new naturally occurring families of interrupted A domains, containing unique architecture and insertion sites. Family 6 interrupted A domains contain an M domain between a6-a7, a site that was previously unknown to withstand insertions.…”

“…, a2–a3 and a8–a9 di-interrupted A domains). 46 Most significantly, we have identified two new naturally occurring families of interrupted A domains, containing unique architecture and insertion sites. Family 6 interrupted A domains contain an M domain between a6–a7, a site that was previously unknown to withstand insertions.…”