Chemical Logic of Peptide Branching by Iterative Nonlinear Nonribosomal Peptide Synthetases

Biochemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 23, 2025

Branch-point syntheses in nonribosomal peptide assembly are rare but useful strategies to generate tripodal peptides with advantageous hexadentate iron-chelating capabilities, as seen siderophores. However, the chemical logic underlying branching by synthetase (NRPS) often remains complex and elusive. Here, we review common for biosynthesis of branched (NRPs) present our biochemical investigation on NRPS-catalyzed fimsbactin A, a mixed-ligand siderophore produced human pathogenic strain Acinetobacter baumannii. We untangled unusual mechanism A through combination bioinformatics, site-directed mutagenesis, vitro reconstitution, molecular modeling, dynamics simulation. Our findings clarify roles NRPS enzymes, uncovering catalytically redundant domains identifying multifunctional nature FbsF cyclization (Cy) domain. demonstrate dynamic interplay between l-serine 2,3-dihydroxybenzoic acid derived dipeptides, partitioning amide ester forms via 1,2-N-to-O-acyl shift orchestrated noncanonical, multichannel Cy The event occurs secondary condensation facilitated this domain two dipeptidyl intermediates, which generates tetrapeptide thioester. Finally, terminal FbsG recruits soluble nucleophile release final product. This study advances understanding intricate biosynthetic pathways employed NRPSs, shedding light mechanisms synthesis peptides.

Language: Английский

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Discovery of a Bacterial Hydrazine Transferase That Constructs the N-Aminolactam Pharmacophore in Albofungin Biosynthesis

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(19), P. 13399 - 13405

Published: May 3, 2024

Structural motifs containing nitrogen-nitrogen (N-N) bonds are prevalent in a large number of clinical drugs and bioactive natural products. Hydrazine (N

Language: Английский

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Conserved Enzymatic Cascade for Bacterial Azoxy Biosynthesis

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(49), P. 27131 - 27139

Published: Nov. 29, 2023

Azoxy compounds exhibit a wide array of biological activities and possess distinctive chemical properties. Although there has been considerable interest in the biosynthetic mechanisms azoxy metabolites, enzymatic basis responsible for bond formation remained largely enigmatic. In this study, we unveil enzyme cascade that constructs valanimycin biosynthesis. Our research demonstrates pair metalloenzymes, comprising membrane-bound hydrazine synthase nonheme diiron synthase, collaborate to convert an unstable pathway intermediate product through hydrazine-azo-azoxy pathway. Additionally, by characterizing homologues from other metabolite pathways, propose two-enzyme could represent conserved strategy bacteria. These findings provide significant mechanistic insights into N–N should facilitate targeted isolation bioactive genome mining.

Language: Английский

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Bacterial Hydrazine Biosynthetic Pathways Featuring Cupin/Methionyl tRNA Synthetase‐like Enzymes

ChemBioChem, Journal Year: 2024, Volume and Issue: 25(9)

Published: March 9, 2024

Nitrogen-Nitrogen (N-N) bond-containing functional groups in natural products and synthetic drugs play significant roles exerting biological activities. The mechanisms of N-N bond formation organic molecules have garnered increasing attention over the decades. Recent advances illuminated various enzymatic nonenzymatic strategies, our understanding construction is rapidly expanding. A group didomain proteins with zinc-binding cupin/methionyl-tRNA synthetase (MetRS)-like domains, also known as hydrazine synthetases, generates amino acid-based hydrazines, which serve key biosynthetic precursors diverse functionalities such hydrazone, diazo, triazene, pyrazole, pyridazinone groups. In this review, we summarize current knowledge on pathways employing unique bond-forming machinery.

Language: Английский

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Revision of the Formycin A and Pyrazofurin Biosynthetic Pathways Reveals Specificity for d-Glutamic Acid and a Cryptic N-Acylation Step During Pyrazole Core Formation

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: March 19, 2025

Formycin A and pyrazofurin are two naturally occurring pyrazole-derived C-nucleosides with antibacterial antiviral activities. While earlier studies have established the chemistry of C-glycosidic bond formation as well subsequent steps in biosynthesis formycin pyrazofurin, how pyrazole ring itself is constructed remains elusive. N-N was previously reported to involve coupling N6-hydroxylated l-lysine l-glutamic acid catalyzed by hydrazine synthetase PyfG, herein PyfG its homologue ForJ shown instead recognize d-glutamate l-glutamate. The product ForJ/PyfG catalysis then releases α-hydrazino d-glutamic upon processing NAD-dependent oxidoreductase ForL. Furthermore, N-acylation an amino ATP-grasp ligase ForM/PyfJ indispensable for recognition FAD-dependent ForR/PyfK perform dehydrogenation Cα-N thereby form a hydrazone intermediate. This work not only demonstrates that correct substrate but also reveals cryptic step assembly core. These results thus provide significant insights into rings rarely seen natural products.

Language: Английский

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Chemical capture of diazo metabolites reveals biosynthetic hydrazone oxidation

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: May 28, 2025

Chemically reactive microbial natural products have enabled therapeutic development 1,2 via their well-established bioactivities including anticancer, 3 antibiotic, 4,5 and antioxidant 6 activities. However, discovery of metabolites is particularly challenging because they may not tolerate traditional bioactivity-guided isolation workflows. 7 Diazo-containing are a subset highly that display potent bioactivity 8-11 enable powerful (bio)synthetic transformations; 12,13 however, instability the diazo group to light, 14,15 heat, 16,17 mild acid, 18 mechanical shock 19 has precluded efficient application. Here, we develop reactivity-based screening approach capture diazo-containing facilitate by mass spectrometry. This workflow revealed two novel products, 4-diazo-3-oxo-butanoic acid diazoacetone, from human lung pathogen Nocardia ninae . Biosynthetic investigations distinct enzymatic logic for formation involving hydrazone oxidation catalyzed metalloenzyme Dob3, biochemical characterization Dob3 suggests promising future applications in biocatalysis. Overall, our work highlights power reactivity-guided strategies identifying facilitating unique transformations.

Language: Английский

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Phylogeny‐guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase‐like Enzymes

ChemBioChem, Journal Year: 2024, Volume and Issue: 25(7)

Published: Feb. 26, 2024

Cupin/methionyl-tRNA synthetase (MetRS)-like didomain enzymes catalyze nitrogen-nitrogen (N-N) bond formation between Nω-hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N-N bonds. While the combination these two building blocks leads creation diverse hydrazine products, full extent their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny-guided genome-mining related pathways consisting enzymes: flavin-dependent Nω-hydroxylating monooxygenases (NMOs) that produce Nω-hydroxylamine precursors cupin/MetRS-like couple with via A phylogenetic analysis identified unexplored sequence spaces enzyme families. The biochemical characterization NMOs demonstrated capabilities Nω-hydroxylamines, including those previously not known as Furthermore, five new novel combinations blocks, one non-amino acid blocks: 1,3-diaminopropane putrescine. This study substantially expanded variety forming mediated by enzymes.

Language: Английский

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Recent Developments and Challenges in the Enzymatic Formation of Nitrogen–Nitrogen Bonds

ACS Catalysis, Journal Year: 2024, Volume and Issue: 15(1), P. 310 - 342

Published: Dec. 17, 2024

The biological formation of nitrogen–nitrogen (N–N) bonds represents intriguing reactions that have attracted much attention in the past decade. This interest has led to an increasing number N–N bond-containing natural products (NPs) and related enzymes catalyze their (referred this review as NNzymes) being elucidated studied greater detail. While more detailed information on biosynthesis NPs, which only become available recent years, provides unprecedented source biosynthetic enzymes, potential for biocatalytic applications been minimally explored. With review, we aim not provide a comprehensive overview both characterized NNzymes hypothetical biocatalysts with putative bond forming activity, but also highlight from perspective. We present compare conventional synthetic approaches linear cyclic hydrazines, hydrazides, diazo- nitroso-groups, triazenes, triazoles allow comparison enzymatic routes via these functional groups. Moreover, pathways well diversity reaction mechanisms are presented according direct groups currently accessible enzymes.

Language: Английский

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Reconstitution of the Final Steps in the Biosynthesis of Valanimycin Reveals the Origin of Its Characteristic Azoxy Moiety

Angewandte Chemie International Edition, Journal Year: 2023, Volume and Issue: 63(1)

Published: Nov. 15, 2023

Valanimycin is an azoxy-containing natural product isolated from the fermentation broth of Streptomyces viridifaciens MG456-hF10. While biosynthesis valanimycin has been partially characterized, how azoxy group constructed remains obscure. Herein, membrane protein VlmO and putative hydrazine synthetase ForJ formycin biosynthetic pathway are demonstrated to catalyze N-N bond formation converting O-(l-seryl)-isobutyl hydroxylamine into N-(isobutylamino)-l-serine. Subsequent installation shown be catalyzed by non-heme diiron enzyme VlmB in a reaction which single VlmO/ForJ oxidized four electrons yield group. The catalytic cycle appears begin with resting μ-oxo diferric complex VlmB, as supported Mössbauer spectroscopy. This study also identifies N-(isobutylamino)-d-serine alternative substrate for leading two regioisomers. reactions kinase VlmJ lyase VlmK during final steps established well. was thus fully reconstituted vitro using enzymes VlmO/ForJ, VlmK. Importantly, VlmB-catalyzed represents first example enzyme-catalyzed expected proceed atypical mechanism.

Language: Английский

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Production of Phenyldiazene Derivatives Using the Biosynthetic Pathway of an Aromatic Diazo Group‐Containing Natural Product from an Actinomycete

ChemBioChem, Journal Year: 2024, Volume and Issue: 26(1)

Published: Oct. 18, 2024

Abstract The diazo group is an important functional in organic synthesis because it confers high reactivity to the compounds and has been applied various chemical reactions, such as Sandmeyer reaction, Wolff rearrangement, cyclopropanation, C−N bond formation with active methylene compounds. Previously, we revealed that 3‐diazoavenalumic acid (3‐DAA), which potentially produced by several actinomycete species contains aromatic group, a biosynthetic intermediate of avenalumic acid. In this study, aimed construct production system for phenyldiazene derivatives adding culture 3‐DAA‐producing recombinant actinomycete. First, acetoacetanilide its derivatives, have are raw materials arylide yellow dyes, were individually added When their metabolites analyzed, each expected compound phenyldiazenyl moiety was detected extract. Moreover, established one‐pot vitro enzymatic same using highly reactive diazotase, CmaA6. These results showed natural products attractive tool expanding structural diversity both vivo .

Language: Английский

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