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

Interrupted adenylation (A) domains are key to the immense structural diversity seen in the nonribosomal peptide (NRP) class of natural products (NPs). Interrupted A domains are A domains that contain within them the catalytic portion of another domain, most commonly a methylation (M) domain. It has been well documented that methylation events occur with extreme specificity on either the backbone (N-) or side chain (O- or S-) of the amino acid (or amino acid-like) building blocks of NRPs. Here, through taxonomic and phylogenetic analyses as well as multiple sequence alignments, we evaluated the similarities and differences between interrupted A domains. We probed their taxonomic distribution amongst bacterial organisms, their evolutionary relatedness, and described conserved motifs of each type of M domain found to be embedded in interrupted A domains. Additionally, we categorized interrupted A domains and the M domains within them into a total of seven distinct families and six different types, respectively. The families of interrupted A domains include two new families, 6 and 7, that possess new architectures. Rather than being interrupted between the previously described a2–a3 or a8–a9 of the ten conserved A domain sequence motifs (a1–a10), family 6 contains an M domain between a6–a7, a previously unknown interruption site. Family 7 demonstrates that di-interrupted A domains exist in Nature, containing an M domain between a2–a3 as well as one between a6–a7, displaying a novel arrangement. These in-depth investigations of amino acid sequences deposited in the NCBI database highlighted the prevalence of interrupted A domains in bacterial organisms, with each family of interrupted A domains having a different taxonomic distribution. They also emphasized the importance of utilizing a broad range of bacteria for NP discovery. Categorization of the families of interrupted A domains and types of M domains allowed for a better understanding of the trends of naturally occurring interrupted A domains, which illuminated patterns and insights on how to harness them for future engineering studies.