Craig A. Townsend

β-Ketoacyl-ACP synthase (KAS) III enzymes play central roles in fatty acid biosynthesis and the production of various natural products. While their canonical function involves catalyzing C–C bond formation between acetyl-CoA and malonyl acyl-carrier-proteins (ACPs), an increasing number of non-canonical activities have been reported among KAS III homologues, including C–O and C–N bond formation. Here, we describe a KAS III family enzyme, CalO4, that catalyzes C–S bond formation during the biosynthesis of the potent antitumor agent calicheamicin. We successfully reconstituted its transacylation activities, demonstrated C–S bond formation, and assessed its substrate selectivity. Furthermore, X-ray crystallography combined with molecular docking and mutational analysis identified key residues likely involved in substrate selection and catalysis. This work not only expands the functional diversity of the KAS III family but also provides a potential biocatalyst for hindered C–S bond formation and a target for engineering novel calicheamicin-like or other bioactive compounds.

S-Adenosyl-l-methionine (SAM) is well-known as a methyl donor for methyltransferases but also functions as a 3-amino-3-carboxypropyl (3-ACP) donor for 3-ACP transferases. NAT is a 3-ACP transferase which accepts β-lactam antibiotic nocardicin G (1) and SAM to produce isonocardicin C. Due to the lack of structural information about this enzyme, its reaction mechanism has not been fully identified. In this study, we report two X-ray crystal structures of NAT, including its apo and complex structure with 1 and SAH. Examination of them identified the structural basis for substrate recognition. Comprehensive approach integrating site-directed mutagenesis, thermal shift assay, MD simulation, and QM/MM calculation revealed that the Cα-amino group of SAM functions as a Brønsted base to enhance the nucleophilicity of the C6'-OH of 1, with the assistance of E143, thereby facilitating S_N2 attack on the Cγ of SAM. This study presents structural and computational analysis leading to more precise understanding of 3-ACP transfer.

Flavin‐dependent halogenases (FDHs) play important roles in natural product biosynthesis, particularly chlorinases and brominases. Apart from ubiquitous mammalian iodination in thyroxine biosynthesis, iodinated natural products are extremely rare, and enzymes responsible for iodination are even less well described. A notable exception is calicheamicin γ ₁ ^I , a potent antitumor compound containing an aromatic iodide, for which a halogenase has been proposed to mediate iodine incorporation. Despite predictions regarding the enzymes involved in iodinated aryl ring biosynthesis, experimental evidence remains limited due to challenges in substrate identification and reaction monitoring. In this study, we successfully reconstituted the enzymatic activities required for iodination and all embellishments of the highly substituted benzene ring in calicheamicin γ ₁ ^I . Using intact‐protein mass spectrometry combined with protease cleavage, we demonstrated formation of the orsellinate thioester followed by sequential C‐2 O ‐methylation, C‐5 iodination, C‐3 oxidation, and C‐3 O ‐methylation. This research characterizes the first flavin‐dependent iodinase that acts on a carrier protein‐dependent substrate, identifies the natural substrates of a cytochrome P450 oxygenase and two O ‐methyltransferases, and provides valuable insights for biocatalysis. Additionally, these findings could facilitate the engineering of other polyketide biosynthetic pathways and contribute to optimizing fermentation conditions to generate new calicheamicin derivatives.

Flavin‐dependent halogenases (FDHs) play important roles in natural product biosynthesis, particularly chlorinases and brominases. Apart from ubiquitous mammalian iodination in thyroxine biosynthesis, iodinated natural products are extremely rare, and enzymes responsible for iodination are even less well described. A notable exception is calicheamicin γ ₁ ^I , a potent antitumor compound containing an aromatic iodide, for which a halogenase has been proposed to mediate iodine incorporation. Despite predictions regarding the enzymes involved in iodinated aryl ring biosynthesis, experimental evidence remains limited due to challenges in substrate identification and reaction monitoring. In this study, we successfully reconstituted the enzymatic activities required for iodination and all embellishments of the highly substituted benzene ring in calicheamicin γ ₁ ^I . Using intact‐protein mass spectrometry combined with protease cleavage, we demonstrated formation of the orsellinate thioester followed by sequential C‐2 O ‐methylation, C‐5 iodination, C‐3 oxidation, and C‐3 O ‐methylation. This research characterizes the first flavin‐dependent iodinase that acts on a carrier protein‐dependent substrate, identifies the natural substrates of a cytochrome P450 oxygenase and two O ‐methyltransferases, and provides valuable insights for biocatalysis. Additionally, these findings could facilitate the engineering of other polyketide biosynthetic pathways and contribute to optimizing fermentation conditions to generate new calicheamicin derivatives.

The simple but essential azetidinone core of the β-lactam antibiotics is uniquely N-sulfonated in the monobactam subfamily. This feature confers both target binding specificity to inactivate bacterial cell wall biosynthesis (antibiosis) and structural differentiation to elude destruction by metallo-β-lactamases (MBLs). The recent FDA approval of Emblaveo to treat serious bacterial infections combines an established synthetic monobactam aztreonam and avibactam, which additionally blocks serine β-lactamases, to create a broadly effective antibacterial therapeutic. Here we report experiments to capture the native monobactam biosynthetic steps to the natural product sulfazecin with the aim of accessing new monobactams by reprogramming its biosynthetic machinery. In sulfazecin biosynthesis, the β-lactam ring is formed by a nonribosomal peptide synthetase SulM that incorporates l-2,3-diaminopropionate (Dap), which is then N-sulfonated in trans and efficiently cyclized to the fully elaborated monobactam by an unusual thioesterase (TE) domain. We describe an improved synthesis of (2S,3R)-vinylDap to support rational structure-based engineering experiments to obtain the corresponding (4R)-vinyl sulfazecin. While these experiments were initially based on an AlphaFold model of the adenylation domain that incorporates Dap (SulM A3), we further report high-resolution X-ray crystal structures with both the l-Dap substrate and an accurate analogue of the activated (3R)-methyl-Dap adenylate bound. The ligand-bound structures rationalize the inability of SulA3 to incorporate larger substrates. Comparisons with the structures of other diamino acid-activating adenylation domains identify alternate binding modes that may be more suitable for the production of sulfazecin analogues. The impact of these structures on the further engineering of the SulA3 domain and its relation to monobactam synthesis in the recently structurally characterized SulTE are discussed.

The enediyne antitumor antibiotics have remarkable structures and exhibit potent DNA cleavage properties that have inspired continued interest as cancer therapeutics. Their complex structures and high reactivity, however, pose formidable challenges to their production and development in the clinic. We report here proof‐of‐concept studies using a mutasynthesis strategy to combine chemical synthesis of select modifications to a key iodoanthracene‐γ‐thiolactone intermediate in the biosynthesis of dynemicin A and all other known anthraquinone‐fused enediynes (AFEs). By chemical complementation of a mutant bacterial producer that is incapable of synthesizing this essential building block, we show that derivatives of dynemicin can be prepared substituted in the A‐ring of the anthraquinone motif. In the absence of competition from native production of this intermediate, the most efficient utilization of these externally‐supplied structural analogues for precursor‐directed biosynthesis becomes possible. To achieve this goal, we describe the required Δorf15 blocked mutant and a general synthetic route to a library of iodoanthracene structural variants. Their successful incorporation opens the door to enhancing DNA binding and tuning the bioreductive activation of the modified enediynes for DNA cleavage.

Enediyne antibiotics are a striking family of DNA-cleaving natural products with high degrees of cytotoxicity and structural complexity. Microbial genome sequences, which have recently accumulated, point to an untapped trove of "cryptic" enediynes. Most of the cognate biosynthetic gene clusters (BGCs) are sparingly expressed under standard growth conditions, making it difficult to characterize their products. Herein, we report a fluorescence-based DNA cleavage assay coupled with high-throughput elicitor screening for the rapid, targeted discovery of cryptic enediyne metabolites. We applied the approach to Streptomyces clavuligerus, which harbors two such BGCs with unknown products, identified steroids as effective elicitors, and characterized 10 cryptic enediyne-derived natural products, termed clavulynes A-J with unusual carbonate and terminal olefin functionalities, with one of these congeners matching the recently reported jejucarboside. Our results contribute to the growing repertoire of enediynes and provide a blueprint for identifying additional ones in the future.

The naturally occurring enediynesare notable for theircomplexstructures, potent DNA cleaving ability, and emerging usefulness incancer chemotherapy. They can be classified into three distinct structuralfamilies, but all are thought to originate from a common linear C-15-heptaene. Dynemicin A (DYN) is the paradigm member of anthraquinone-fusedenediynes, one of the three main classes and exceptional among themfor derivation of both its enediyne and anthraquinone portions fromthis same early biosynthetic building block. Evidence is growing abouthow two structurally dissimilar, but biosynthetically related, intermediatescombine in two heterodimerization reactions to create a nitrogen-containingC(30)-coupled product. We report here deletions of two genesthat encode biosynthetic proteins that are annotated as S-adenosylmethionine (SAM)-dependent methyltransferases. While one,DynO6, is indeed the required O-methyltransferaseimplicated long ago in the first studies of DYN biosynthesis, theother, DynA5, functions in an unanticipated manner in the post-heterodimerizationevents that complete the biosynthesis of DYN. Despite its removalfrom the genome of Micromonospora chersina, the Delta dynA5 strain retains the ability tosynthesize DYN, albeit in reduced titers, accompanied by two unusualco-metabolites. We link the appearance of these unexpected structuresto a substantial and contradictory body of other recent experimentaldata to advance a biogenetic rationale for the downstream steps thatlead to the final formation of DYN. A sequence of product-formingtransformations that is in line with new and existing experimentalresults is proposed and supported by a model reaction that also encompassesthe formation of the crucial epoxide essential for the activationof DYN for DNA cleavage.