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Interrupted adenylation (A) domains are fascinating examples of multifunctional enzymes. They are found in nonribosomal peptide synthetases (NRPSs), which biosynthesize nonribosomal peptides (NRPs), a major class of medically relevant natural products (NPs). Interrupted A domains contain the catalytic portion of another domain within them, typically a methylation (M) domain, thus combining both adenylation and methylation capabilities. In recent years, interrupted A domains have demonstrated tremendous enzyme engineering potential as they are able to be constructed artificially in a laboratory setting by combining the A and M domains of two separate NRPS proteins. A recent discovery and characterization of a naturally occurring interrupted A domain that harbored two M domains back-to-back, a trifunctional protein, showed the ingenuity of Nature to both N - and O -methylate amino acids, the building blocks of NRPs. Since we have shown that a single M domain could be added to an uninterrupted A domain to create an artificial interrupted A domain, we set out to investigate if: (i) an A domain could be engineered to contain two back-to-back M domains and (ii) the added M domains would have to reflect the pattern in Nature, a side chain ( O -) methylating M domain (M s ) followed by a backbone ( N -) methylating M domain (M b ), or if the order of the M domains could be reversed. To address these questions, we set out to create our own AM s M b A and AM b M s A engineered interrupted A domains. We evaluated these engineered proteins connected (in cis ) and/or disconnected (in trans ) from the native thiolation (T) domain, through a series of radiometric assays, high performance liquid chromatography (HPLC), and mass spectrometry (MS) for adenylation, loading, and methylation ability. We found that although adenylation activity was preserved in both versions (AM s M b A and AM b M s A), addition of the M domains, in natural and unnatural order, did not result in the desired added methylation capability. This study offers valuable insights into the limits of constructing engineered interrupted A domains as potential tools for modifications of NRPs.

rsc advances

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