The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain.

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain that catalyzes an unusual ß-lactam forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the ß-lactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The structure illustrates a constrained active site that orients the substrate properly for ß-lactam formation. In this regard, the structure is similar to the ß-lactone forming thioesterase domain responsible for the production of obafluorin. Analysis of the structure identifies features that are responsible for this four-membered ring closure and enable bioinformatic analysis to identify additional, uncharacterized ß-lactam-forming biosynthetic gene clusters by genome mining.

Erratum: The dabABC operon is a marker of C4-alkylated monobactam biosynthesis and responsible for (2S,3R)-diaminobutyrate production.

[This corrects the article DOI: 10.1016/j.isci.2024.109202.].

The dabABC operon is a marker of C4-alkylated monobactam biosynthesis and responsible for (2S,3R)-diaminobutyrate production.

Non-ribosomal peptide synthetases (NRPSs) assemble metabolites of medicinal and commercial value. Both serine and threonine figure prominently in these processes and separately can be converted to the additional NRPS building blocks 2,3-diaminopropionate (Dap) and 2,3-diaminobutyrate (Dab). Here we bring extensive bioinformatics, in vivo and in vitro experimentation to compose a unified view of the biosynthesis of these widely distributed non-canonical amino acids that both derive by pyridoxal-mediated ß-elimination of the activated O-phosphorylated substrates followed by ß-addition of an amine donor. By examining monobactam biosynthesis in Pseudomonas and in Burkholderia species where it is silent, we show that (2S,3R)-Dab synthesis depends on an l-threonine kinase (DabA), a ß-replacement reaction with l-aspartate (DabB) and an argininosuccinate lyase-like protein (DabC). The growing clinical importance of monobactams to both withstand Ambler Class B metallo-ß-lactamases and retain their antibiotic activity make reprogrammed precursor and NRPS synthesis of modified monobactams a feasible and attractive goal.

Targeted Discovery of Cryptic Enediyne Natural Products via FRET-Coupled High-Throughput Elicitor Screening.

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.

Normal and Aberrant Methyltransferase Activities Give Insights into the Final Steps of Dynemicin A Biosynthesis.

The naturally occurring enediynes are notable for their complex structures, potent DNA cleaving ability, and emerging usefulness in cancer chemotherapy. They can be classified into three distinct structural families, but all are thought to originate from a common linear C15-heptaene. Dynemicin A (DYN) is the paradigm member of anthraquinone-fused enediynes, one of the three main classes and exceptional among them for derivation of both its enediyne and anthraquinone portions from this same early biosynthetic building block. Evidence is growing about how two structurally dissimilar, but biosynthetically related, intermediates combine in two heterodimerization reactions to create a nitrogen-containing C30-coupled product. We report here deletions of two genes that encode biosynthetic proteins that are annotated as S-adenosylmethionine (SAM)-dependent methyltransferases. While one, DynO6, is indeed the required O-methyltransferase implicated long ago in the first studies of DYN biosynthesis, the other, DynA5, functions in an unanticipated manner in the post-heterodimerization events that complete the biosynthesis of DYN. Despite its removal from the genome of Micromonospora chersina, the ΔdynA5 strain retains the ability to synthesize DYN, albeit in reduced titers, accompanied by two unusual co-metabolites. We link the appearance of these unexpected structures to a substantial and contradictory body of other recent experimental data to advance a biogenetic rationale for the downstream steps that lead to the final formation of DYN. A sequence of product-forming transformations that is in line with new and existing experimental results is proposed and supported by a model reaction that also encompasses the formation of the crucial epoxide essential for the activation of DYN for DNA cleavage.

Synthesis of functionalized 2,3-diaminopropionates and their potential for directed monobactam biosynthesis.

The N-sulfonated monobactams harbor considerable potential to combat emerging bacterial infections that are problematic to treat due to their metallo-ß-lactamase mediated resistance against conventional ß-lactam antibiotics. Herein, we report a divergent synthesis of C3-substituted 2,3-diaminopropionates featuring an array of small functional groups and examine their potential as alternative precursors during monobactam biosynthesis in a mutant strain (ΔsulG) of Pseudomonas acidophila that is deficient in the supply of this native precursor. In vitro assays revealed high diastereoselectivity, as well as a substrate tolerance by the terminal adenylation domain of the non-ribosomal peptide synthetase (NRPS) SulM toward the majority of synthetic analogs. Chemical complementation of this mutant yielded a fluorinated, bioactive monobactam through fermentation as confirmed by a combination of spectrometric data and microbiological assays. This study demonstrates site-specific functionalization of a clinically important natural product and sets in place a platform for further strain improvements and engineered NRPS-biosynthesis of non-native congeners.

Examining Heterodimerization by Aryl C-N Coupling in Dynemicin Biosynthesis.

Distinct among the enediyne antitumor antibiotics, the dynemicin subgroup is comprised of two discrete halves, an enediyne and an anthraquinone, but each is ultimately derived from the same linear ß-hydroxyhexaene precursor. The linkage of these two halves by an aryl C-N bond is examined here using a variety of experimental approaches. We demonstrate that this heterodimerization is specific for anthracenyl iodide as the corresponding bromo- and amino-substituted anthracenes do not support dynemicin biosynthesis. Furthermore, biochemical experiments and chemical model reactions support an SRN1 mechanism for the aryl C-N coupling in which electron transfer occurs to the iodoanthracene, followed by loss of an anthracenyl iodide and partition of the resulting aryl radical between C-N coupling and reduction by hydrogen abstraction. An enzyme pull-down experiment aiming to capture the protein(s) involved in the coupling reaction is described in which two proteins, Orf14 and Orf16, encoded by the dynemicin biosynthetic gene cluster, are specifically isolated. Deletion of orf14 from the genome abolished dynemicin production accompanied by a 3-fold increased accumulation of the iodoanthracene coupling partner, indicating the plausible involvement of this protein in the heterodimerization process. On the other hand, the deletion of orf16 only reduced dynemicin production to 55%, implying a noncatalytic, auxiliary role of the protein. Structural comparisons using AlphaFold imply key similarities between Orf14 and X-ray crystal structures of several proteins from enediyne BGCs believed to bind hydrophobic polyene or enediyne motifs suggest Orf14 templates aryl C-N bond formation during the central heterodimerization in dynemicin biosynthesis.

Colorimetric Determination of Adenylation Domain Activity in Nonribosomal Peptide Synthetases by Using Chrome Azurol S.

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.

ThnL, a B12-dependent radical S-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.

Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.

Structure-Activity Relationship of Penem Antibiotic Side Chains Used against Mycobacteria Reveals Highly Active Compounds.

The rise of antibiotic-resistant Mycobacterium tuberculosis and non-tuberculous mycobacterial infections has placed ever-increasing importance on discovering new antibiotics to treat these diseases. Recently, a new penem, T405, was discovered to have strong antimicrobial activity against M. tuberculosis and Mycobacteroides abscessus. Here, a penem library of C2 side-chain variants was synthesized, and their antimicrobial activities were evaluated against M. tuberculosis H37Rv and M. abscessus ATCC 19977. Several new penems with antimicrobial activity stronger than the standard-of-care carbapenem antibiotics were identified with some candidates improving on the activity of the lead compound, T405. Moreover, many candidates showed little or no increase in the minimum inhibitory concentration in the presence of serum compared to the highly protein-bound T405. The penems with the strongest activity identified in this study were then biochemically characterized by reaction with the representative l,d-transpeptidase LdtMt2 and the representative penicillin-binding protein d,d-carboxypeptidase DacB2.

Accurate Substrate-Like Probes for Trapping Late-Stage Intermediates in Nonribosomal Peptide Synthetase Condensation Domains.

Nonribosomal peptide synthetases (NRPSs) are a family of multidomain enzymes dedicated to the production of peptide natural products. Central to NRPS function are condensation (C) domains, which catalyze peptide bond formation and a number of specialized transformations including dehydroamino acid and ß-lactam synthesis. Structures of C domains in catalytically informative states are limited due to a lack of clear strategies for stabilizing C domain interactions with their substrates and client domains. Inspired by a ß-lactam forming C domain, we report herein the synthesis and application of 1, which forms irreversible cross-links with engineered thiol nucleophiles in a C domain active site. Deployment of 1 demonstrates the synthetic tractability of trapping late-stage nascent peptides in C domains and provides a readily adaptable tactic for stabilizing C domain interactions in multidomain NRPS fragments.

Stereochemical course of cobalamin-dependent radical SAM methylation by TokK and ThnK.

Complex carbapenems are important clinical antibiotics for difficult-to-treat infections. An essential step in the biosyntheses of these natural products is stereospecific methylation at C6 and subsequent alkylations by cobalamin-dependent radical SAM methylases such as TokK and ThnK. We have prepared isotopically labeled substrates in a stereospecific manner and found that both homologous enzymes selectively abstract the 6-pro-S hydrogen, followed by methyl transfer to the opposite face to give the (6R)-methyl carbapenam product proceeding, therefore, by inversion of absolute configuration at C6. These data clarify an unexpected ambiguity in the recently solved substrate-bound crystal structure of TokK and have led to a stereochemically complete mechanistic proposal for both TokK and ThnK.

Purification and characterization of sequential cobalamin-dependent radical SAM methylases ThnK and TokK in carbapenem ß-lactam antibiotic biosynthesis.

ThnK and TokK are cobalamin-dependent radical S-adenosylmethionine enzymes that catalyze sequential methylations of a common carbapenem biosynthetic intermediate. ThnK was an early characterized member of the subfamily of cobalamin-dependent radical S-adenosylmethionine enzymes. Since initial publication of the ThnK function, the field has progressed, and we have made methodological strides in the expression and purification of this enzyme and its ortholog TokK. An optimized protocol for obtaining the enzymes in pure and active form has enabled us to characterize their reactions and gain greater insight into the kinetic behavior of the sequential methylations they catalyze. We share here the methods and strategy that we have developed through our study of these enzymes.

T405, a New Penem, Exhibits In Vivo Efficacy against M. abscessus and Synergy with ß-Lactams Imipenem and Cefditoren.

Mycobacteroides abscessus (Mab) is an emerging environmental microbe that causes chronic lung disease in patients with compromised lung function such as cystic fibrosis and bronchiectasis. It is intrinsically resistant to most antibiotics, therefore there are only few antibiotics that can be repurposed to treat Mab disease. Although current recommendations require daily intake of multiple antibiotics for more than a year, cure rate is low and often associated with significant adverse events. Here, we describe in vivo efficacy of T405, a recently discovered ß-lactam antibiotic of the penem subclass, in a mouse model of pulmonary Mab infection. Imipenem, one of the standard-of-care drugs to treat Mab disease, and also a ß-lactam antibiotic from a chemical class similar to T405, was included as a comparator. Probenecid was included with both T405 and imipenem to reduce the rate of their renal clearance. T405 exhibited bactericidal activity against Mab from the onset of treatment and reduced Mab lung burden at a rate similar to that exhibited by imipenem. The MIC of T405 against Mab was unaltered after 4 weeks of exposure to T405 in the lungs of mice. Using an in vitro assay, we also demonstrate that T405 in combination with imipenem, cefditoren or avibactam exhibits synergism against Mab. Additionally, we describe a scheme for synthesis and purification of T405 on an industrial scale. These attributes make T405 a promising candidate for further preclinical assessment to treat Mab disease.

Sea Urchin Polyketide Synthase SpPks1 Produces the Naphthalene Precursor to Echinoderm Pigments.

Nearly every animal species on Earth contains a unique polyketide synthase (PKS) encoded in its genome, yet no animal-clade PKS has been biochemically characterized, and even the chemical products of these ubiquitous enzymes are known in only a few cases. The earliest animal genome-encoded PKS gene to be identified was SpPks1 from sea urchins. Previous genetic knockdown experiments implicated SpPks1 in synthesis of the sea urchin pigment echinochrome. Here, we express and purify SpPks1, performing biochemical experiments to demonstrate that the sea urchin protein is responsible for the synthesis of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (ATHN). Since ATHN is a plausible precursor of echinochromes, this result defines a biosynthetic pathway to the ubiquitous echinoderm pigments and rewrites the previous hypothesis for echinochrome biosynthesis. Truncation experiments showed that, unlike other type I iterative PKSs so far characterized, SpPks1 produces the naphthalene core using solely ketoacylsynthase (KS), acyltransferase, and acyl carrier protein domains, delineating a unique class of animal nonreducing aromatic PKSs (aPKSs). A series of amino acids in the KS domain define the family and are likely crucial in cyclization activity. Phylogenetic analyses indicate that SpPks1 and its homologs are widespread in echinoderms and their closest relatives, the acorn worms, reinforcing their fundamental importance to echinoderm biology. While the animal microbiome is known to produce aromatic polyketides, this work provides biochemical evidence that animals themselves also harbor ancient, convergent, dedicated pathways to carbocyclic aromatic polyketides. More fundamentally, biochemical analysis of SpPks1 begins to define the vast and unexplored biosynthetic space of the ubiquitous animal PKS family.

Evolution of Methods for the Study of Cobalamin-Dependent Radical SAM Enzymes.

While bioinformatic evidence of cobalamin-dependent radical S-adenosylmethionine (SAM) enzymes has existed since the naming of the radical SAM superfamily in 2001, none were biochemically characterized until 2011. In the past decade, the field has flourished as methodological advances have facilitated study of the subfamily. Because of the ingenuity and perseverance of researchers in this field, we now have functional, mechanistic, and structural insight into how this class of enzymes harnesses the power of both the cobalamin and radical SAM cofactors to achieve catalysis. All of the early characterized enzymes in this subfamily were methylases, but the activity of these enzymes has recently been expanded beyond methylation. We anticipate that the characterized functions of these enzymes will become both better understood and increasingly diverse with continued study.

Penicillin Binding Proteins and ß-Lactamases of Mycobacterium tuberculosis: Reexamination of the Historical Paradigm.

Penicillin binding proteins (PBPs) have been extensively studied due to their importance to the physiology of bacterial cell wall peptidoglycan and as targets of the most widely used class of antibiotics, the ß-lactams. The existing paradigm asserts that PBPs catalyze the final step of peptidoglycan biosynthesis, and ß-lactams inhibit their activities. According to this paradigm, a distinct enzyme class, ß-lactamases, exists to inactivate ß-lactams. This paradigm has been the basis for how bacterial diseases are treated with ß-lactams. We tested whether this historical view accurately reflects the relationship between ß-lactams and the PBPs and the ß-lactamase, BlaC, of Mycobacterium tuberculosis. BlaC was the major inactivator of the cephalosporin subclass of ß-lactams. However, the PBPs PonA1 and PonA2 inactivated penicillins and carbapenems more effectively than BlaC. These findings demonstrate that select M. tuberculosis PBPs are effective at inactivating several ß-lactams. Lesser-known PBPs, DacB, DacB1, DacB2, and Rv2864c, a putative PBP, were comparably more resistant to inhibition by all ß-lactam subclasses. Additionally, Rv1730c exhibited low affinity to most ß-lactams. Based on these findings, we conclude that in M. tuberculosis, BlaC is not the only source of inactivation of ß-lactams. Therefore, the historical paradigm does not accurately describe the relationship between ß-lactams and M. tuberculosis. IMPORTANCE M. tuberculosis, the causative agent of tuberculosis, kills more humans than any other bacterium. ß-lactams are the most widely used class of antibiotics to treat bacterial infections. Unlike in the historical model that describes the relationship between ß-lactams and M. tuberculosis, we find that M. tuberculosis penicillin binding proteins are able to inactivate select ß-lactams with high efficiency.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

Competing off-loading mechanisms of meropenem from an l,d-transpeptidase reduce antibiotic effectiveness.

The carbapenem family of ß-lactam antibiotics displays a remarkably broad spectrum of bactericidal activity, exemplified by meropenem's phase II clinical trial success in patients with pulmonary tuberculosis, a devastating disease for which ß-lactam drugs historically have been notoriously ineffective. The discovery and validation of l,d-transpeptidases (Ldts) as critical drug targets of bacterial cell-wall biosynthesis, which are only potently inhibited by the carbapenem and penem structural classes, gave an enzymological basis for the effectiveness of the first antitubercular ß-lactams. Decades of study have delineated mechanisms of ß-lactam inhibition of their canonical targets, the penicillin-binding proteins; however, open questions remain regarding the mechanisms of Ldt inhibition that underlie programs in drug design, particularly the optimization of kinetic behavior and potency. We have investigated critical features of mycobacterial Ldt inhibition and demonstrate here that the covalent inhibitor meropenem undergoes both reversible reaction and nonhydrolytic off-loading reactions from the cysteine transpeptidase LdtMt2 through a high-energy thioester adduct. Next-generation carbapenem optimization strategies should minimize adduct loss from unproductive mechanisms of Ldt adducts that reduce effective drug concentration.

Acyl Donor Stringency and Dehydroaminoacyl Intermediates in ß-Lactam Formation by a Non-ribosomal Peptide Synthetase.

Condensation (C) domains in non-ribosomal peptide synthetases catalyze peptide elongation steps whereby activated amino acid or peptidyl acyl donors are coupled with specific amino acid acceptors. In the biosynthesis of the ß-lactam antibiotic nocardicin A, an unusual C domain converts a seryl tetrapeptide into its pentapeptide product containing an integrated ß-lactam ring. While indirect evidence for the intermediacy of a dehydroalanyl species has been reported, here we describe observation of the elusive enzyme-bound dehydroamino acyl intermediate generated from the corresponding allo-threonyl tetrapeptide and partitioned into pentapeptide products containing either a dehydrobutyrine residue or an embedded ß-lactam. Contrary to trends in the literature where condensation domains have been deemed flexible as to acyl donor structure, this ß-lactam synthesizing domain is highly discriminating. The observation of dehydrobutyrine formation links this C domain to related clades associated with natural products containing dehydroamino acid and d-configured residues, suggesting a common mechanistic link.