Aug 2026· Journal of the American Chemical Society· Vol 148 32, pp.
34895-34906
· 0 citations· 49 references
Medicine
TL;DR
In vitro and in vivo analyses support a recent finding that the defining carbon-carbon bond is formed not by a long-hypothesized PLP-dependent process, but by a vitamin B12-dependent radical SAM enzyme, and expand upon the current knowledge of radical-mediated C-C bond formation and PEARL enzyme catalysis.
Abstract
Sinefungin is a potent nucleoside antimetabolite of S-adenosylmethionine (SAM). Since its discovery in the 1970s, sinefungin has generated significant scientific interest owing to its role as a bioisostere of SAM and its broad range of biological activities. Despite considerable efforts to uncover the enzymes responsible for sinefungin production in the following years, its biosynthesis remained unclear for decades. Here, we characterize the complete sinefungin biosynthetic gene cluster (sin BGC) from Streptomyces incarnatus NRRL 8089. In vitro and in vivo analyses support a recent finding that the defining carbon-carbon (C-C) bond is formed not by a long-hypothesized PLP-dependent process, but by a vitamin B12-dependent radical SAM enzyme. We provide direct mechanistic evidence, via isotope-labeled products, that the adenosyl group of sinefungin originates from adenosylcobalamin and is atypically consumed via a homolytic SH2 substitution reaction. We also characterize two peptide aminoacyl-tRNA ligases (PEARLs) that append alanines onto the nucleoside scaffold using tRNA-activated amino acids. The PEARLs act directly on small molecules rather than macromolecular substrates, with one PEARL capable of iterative elongation. In addition, we perform in-vitro substrate profiling of several sin BGC-encoded enzymes. We reveal that multiple enzymes show specificity toward phosphorylated intermediates, including the earliest-acting PEARL enzyme. These observations provide an explanation for a cryptic phosphorylation-dephosphorylation strategy observed in the pathway, as they prevent the formation of the highly toxic sinefungin inside the cell. Finally, we leverage these enzymes in a reduced multi-enzyme cascade to biosynthesize sinefungin. Together, these findings expand upon our current knowledge of radical-mediated C-C bond formation and PEARL enzyme catalysis, unlocking biocatalytic possibilities to produce amino acid-nucleoside conjugates.
Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that form complex bioactive scaffolds through extensive enzymatic tailoring. The polyglycosylated thiopeptides persiathiacins, exhibit potent activity against multidrug-resistant Mycobacterium tuberculosis (Mtb) and methicillin-resistant Staphylococcus aureus (MRSA). The persiathiacin biosynthetic gene cluster encodes six cytochrome P450 (CYP) enzymes, but the logic of their oxidative modifications was unknown. Here, we establish a protoplast-based genetic system for Actinokineospora and systematically assign functions to all P450s. We demonstrate that PerX hydroxylates the central thiazole, PerV installs the third indole–core crosslink required for macrocyclization, and PerT, not PerU, catalyses indole N-hydroxylation. Combined gene inactivation and metabolite profiling reveal a hierarchical enzymatic sequence leading to the mature scaffold prior to sugar installation. Notably, the intermediate accumulating in the ΩperX mutant exhibits enhanced anti-M. tuberculosis potency compared to persiathiacin A (IC50 = 0.07 vs 1.5 µg mL−1). These results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.
F. A. Sumang, Maxwell T. Stevens, W. Britton et al.· bioRxiv· 0 citations
Peloruside A, a polyketide macrolide identified in the marine sponge Mycale hentscheli, is a promising anticancer drug candidate due to its ability to stabilize tubulin at a nontaxoid binding site. Substantial efforts were made to improve the supply of this scarce substance through chemical synthesis and mariculture, however, sustainable production has yet to be achieved. Recent microbiome sequencing suggested that bacterial producers are responsible for all M. hentscheli bioactive compounds, but the identity of the peloruside producer remained unknown. Furthermore, a candidate peloruside polyketide synthase (PKS) was identified in silico, but its highly aberrant architecture prevented a confident biosynthetic prediction for the pharmacologically important exocyclic double bond. Here, we reveal an unusually complex modular PKS pathway comprising nonelongating ketosynthases (KSs), two internal thioesterases (TEs), and an acetyl coenzyme A ligase that orchestrate O-acetylation/elimination, acetyl-CoA recycling, and configurational proof-reading to facilitate selective Z-double bond incorporation. The first internal TE falls into a previously characterized O-acylating TE family but contains key amino acid substitutions that facilitate bifunctional acylation/elimination activity. Furthermore, we identified the peloruside producer as 'Candidatus Pelorusia occulta', a member of the biochemically underexplored Lentisphaeria class within the phylum Verrucomicrobiota. This work provides the biochemical and microbiological foundation for heterologous peloruside production.
Coumarins and their derivatives possess diverse bioactivities and broad applications in pharmaceuticals, food additives, and materials science. For example, amino-functionalized coumarin derivatives hold significant promise for the development of novel functional materials and drug discovery. However, the microbial biosynthesis of these compounds remains underexplored. In this study, we characterized the substrate flexibility of the type III polyketide synthase PqsD from Pseudomonas aeruginosa in Pseudomonas putida KT2440, showing its ability to convert various para- and meta-substituted salicylic acids into novel coumarin derivatives. Leveraging the substrate promiscuity of PqsD, we established a novel microbial route to produce 6-amino-4-hydroxycoumarin (6A4HC). Furthermore, we developed a new de novo biosynthetic route for its direct precursor, 5-aminosalicylic acid (5ASA), which is an FDA-approved pharmaceutical compound. This work expands the enzymatic toolbox for coumarin biosynthesis and highlights microbial platforms as sustainable alternatives for generating structurally diverse and functionalized coumarins.
The global rise of drug-resistant Mycobacterium tuberculosis (Mtb) underscores an urgent need for antitubercular agents with novel targets and mechanisms of action. Among these, the de novo purine biosynthesis pathway is essential for Mtb growth and survival, making its constituent enzymes attractive targets for therapeutic intervention. Within this pathway, adenylosuccinate (ADS) synthetase (ADSS) Rv0357c catalyzes the first committed step in biosynthesis of adenosine monophosphate (AMP) by converting inosine monophosphate (IMP) to ADS through a GTP-dependent reaction with l-aspartate. Despite its importance, Mtb ADSS remains poorly characterized at the biochemical level. In this study, we report the expression, purification, and enzymatic characterization of recombinant Mtb ADSS. To overcome the challenge of the enzyme being predominantly expressed as inclusion bodies in Escherichia coli, we established both protein refolding and chaperone-assisted expression strategies to obtain soluble, catalytically active protein. Using complementary spectrophotometric, colorimetric, and fluorescence-based assays, we determined steady-state kinetic parameters and confirmed robust ADSS activity consistent with Michaelis-Menten behaviour. Furthermore, we developed scalable, nonradioactive assays compatible with high-throughput screening (HTS), enabling the quantitative monitoring of ADSS activity via GTP hydrolysis and phosphate release. As a proof of concept, the MESG assay successfully detected inhibition of Mtb ADSS by the previously reported ADSS inhibitor Aurodox, demonstrating its utility for inhibitor characterization and screening. Collectively, these results provide the first comprehensive biochemical framework for studying Mtb ADSS and establish a foundation for structure-guided inhibitor discovery targeting purine biosynthesis as a novel antitubercular strategy.
Vigyasa Singh, Ran Zhang, Ke Chen et al.· Biochimica et Biophysica Act...· 0 citations
Lanthipeptides are one of the largest classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). The coi biosynthetic gene cluster (BGC) from Streptomyces coelicolor A3(2) encodes a canonical class I lanthipeptide dehydratase (CoiB) and cyclase (CoiC), a bifunctional enzyme (CoiSA) with an O-methyltransferase (MT) and glutamyl lyase (GL) domain, and a protein of unknown function (CoiH). The product of the coi BGC was recently shown to impart anti-phage activity, but its structure is still unresolved. Previous work investigated the regioselectivity of the GL domains in CoiB and CoiSA and the stereochemistry of the cyclized precursor peptide, but the function of CoiH was not addressed. In this study, co-expression of the peptide CoiA1 with CoiBCSAH resulted in a +16 Da addition on the cyclized peptide compared to when CoiH was omitted. LC-MS/MS analysis indicated that this modification occurred in the first thioether ring. A combination of site-directed mutagenesis, comparison of linear and cyclized peptide substrates, hydrogen peroxide (H₂O₂) treatment, and collision-induced dissociation (CID) mass spectrometric analysis suggested that the sulfur atom in the first methyllanthionine was oxidized to a sulfoxide group by CoiH. This hypothesis was confirmed by NMR analysis. CoiH represents a previously uncharacterized oxygenase family catalyzing sulfoxide formation. Structure prediction tools suggest a novel enzyme fold without obvious metal or cofactor binding sites, raising the possibility that CoiH is a cofactor independent oxidation enzyme.
Jingqi Chen, Lingyang Zhu, W. A. van der Donk· bioRxiv· 0 citations
Fungal diseases are an escalating threat to human health, driven by rising antimicrobial resistance and a lack of new antifungal treatments1,2. Polyenes, a class of complex natural products, have been widely used as antifungal agents due to their broad-spectrum activity3. However, their clinical use is limited by severe toxicity and poor solubility, making safer and more effective alternatives urgently needed4. Previous efforts to generate improved polyenes have relied on costly, step-inefficient and atom-inefficient chemical syntheses5,6. Here we describe the discovery and characterization of pathways to previously undescribed polyenes, including unusual glycosyltransferase enzymes that introduce sugars onto polyene scaffolds. We also show how the reaction scope of an amidotransferase enzyme can be expanded to transform the detrimental carboxylate substituent of polyenes to alternative functionality. Introduction of a second sugar combined with carboxylate modifications leads to more potent polyene antifungals, with reduced toxicity, that are accessible by clean and efficient fermentation or enzymatic routes. Pathways to previously undescribed polyenes, including unusual enzymes that introduce more sugars onto polyene scaffolds, are discovered, and their accessibility through clean and efficient fermentation and potential for safe use as antifungal treatments is described.
Saadia N. Mirza, Alberto Carella, Joseph W. Thompson et al.· Nature· 1 citation
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