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.

Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature N,S-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS(AMAT) and TioN(AMA). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for N,S-dimethylation of l-Cys, but also revealed that a single interrupted A domain can N,N-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. These findings offer important and useful insights for the development and engineering of novel interrupted A domain enzymes to serve, in the future, as tools for combinatorial biosynthesis.

Controlled halogenation of chemically versatile substrates is difficult to achieve. Here we describe a unique flavin-dependent halogenase, PltM, which is capable of utilizing a wide range of halides for installation on a diverse array of phenolic compounds, including FDA-approved drugs and natural products, such as terbutaline, fenoterol, resveratrol, and catechin. Crystal structures of PltM in complex with phloroglucinol and FAD in different states yield insight into substrate recognition and the FAD recycling mechanism of this halogenase.

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(M) and TioS(M), into a naturally occurring uninterrupted A domain, Ecm6(AT). We evaluated the engineered enzymes, Ecm6(AMAT) and Ecm6(AMAT), 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.

Only a handful of aziridine-containing natural products have been identified out of the more than 100,000 natural products characterized to date. Among this class of compounds, only the azinomycins (azinomycin A and B) and ficellomycin contain an unusual 1-azabicyclo[3.1.0]hexane ring system, which has been reported to be the reason for theDNAcrosslinking abilities and cytotoxicity of these metabolites. Both families of natural products are produced by Streptomyces species, Streptomyces sahachiroi and Streptomyces ficellus, respectively. Up until recently, much of the work on these molecules has focused on the synthesis of these natural products or their corresponding analogs for in vitro investigations evaluating their DNA selectivity. While one of the most intriguing aspects of these natural products is their biosynthesis, progress made in this area was largely impeded by difficulties with obtaining a reliable culture method and securing a consistent source of these natural products. In this review, we will cover the discovery and biological activity of the azinomycins, their mode of action, related synthetic analogs and biosynthesis, and finish with a discussion on the less studied metabolite, ficellomycin.

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(AM s M b A). 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.

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

A new bioactive macrolactone, nuiapolide (1) was identified from a marine cyanobacterium collected off the coast of Niihau, near Lehua Rock. The natural product exhibits anti-chemotactic activity at concentrations as low as 1.3 μM against Jurkat cells, cancerous T lymphocytes, and induces a G2/M phase cell cycle shift. Structural characterization of the natural product revealed the compound to be a 40-membered macrolactone with nine hydroxyl functional groups and a rare tert-butyl carbinol residue.