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

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 (Ms) followed by a backbone (N-) methylating M domain (Mb), or if the order of the M domains could be reversed. To address these questions, we set out to create our own AMsMbA and AMbMsA 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 (AMsMbA and AMbMsA), 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.

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.

Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions.

Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.

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

The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M3S domain from TioS(A3aM3SA3b) between the a8-a9 region of a mono-interrupted A domain, TioN(AaMNAb), that already contained a side chain methylating MN domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AMNAM3SA) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.

Structural basis for backbone N-methylation by an interrupted adenylation domain.

Interrupted adenylation domains are enigmatic fusions, in which one enzyme is inserted into another to form a highly unusual bifunctional enzyme. We present the first crystal structure of an interrupted adenylation domain that reveals a unique embedded methyltransferase. The structure and functional data provide insight into how these enzymes N-methylate amino acid precursors en route to nonribosomal peptides.

Engineering Bifunctional Enzymes Capable of Adenylating and Selectively Methylating the Side Chain or Core of Amino Acids.

Nonribosomal peptides (NRPs) are known sources of therapeutics. Some nonribosomal peptide synthetase assembly lines contain unique functional interrupted adenylation (A) domains, where nature has combined two different functional domains into one bifunctional enzyme. Most often these interrupted A domains contain a part of a methylation (M) domain embedded in their sequence. Herein, we aimed to emulate nature and create fully functional interrupted A domains by inserting two different noncognate M domains, KtzH(MH) and TioS(M3S), into a naturally occurring uninterrupted A domain, Ecm6(A1T1). We evaluated the engineered enzymes, Ecm6(A1aMHA1bT1) and Ecm6(A1aM3SA1bT1), by a series of radiometric assays and found that not only do they maintain A domain activity, but also they gain the site-specific methylation patterns observed in the parent M domain donors. These findings provide an exciting proof-of-concept for generating interrupted A domains as future tools to modify NRPs and increase the diversity and activity of potential therapeutics.