Aug 2026· Journal of the American Chemical Society· 0 citations· 31 references
Abstract
Nucleoside natural products exhibit diverse chemical architectures and potent biological activities, yet the biosynthetic strategies that generate their structural diversity remain incompletely understood. Here, we elucidate the early stage biosynthetic pathway of the cytidyl–cyclitol natural product K-563 and its derivatives. The cyclitol component is generated by the myo-inositol-1-phosphate synthase (MIPS) family enzyme KesM and is subsequently coupled to the cytidine moiety by KesL using cytidine 5′-triphosphate (CTP), followed by further modifications catalyzed by the phosphatase KesI and the unique dehydrogenase complex KesJ/KesK. The X-ray crystal structure and mutagenesis analyses reveal that KesL adopts the canonical fold of S-adenosyl-l-methionine (SAM) synthetases, which catalyze the adenosylation of l-methionine with adenosine 5′-triphosphate (ATP) in primary metabolism, while the KesL active site is extensively remodeled to accept the cyclitol phosphate with CTP as an atypical substrate pair, thereby generating the cytidyl–cyclitol core structure. This work not only expands the chemical logic of nucleoside biosynthesis but also demonstrates how the SAM synthetase-like protein scaffold is repurposed to catalyze an unusual nucleoside transfer reaction for specialized secondary metabolite assembly.
Cyanogramide (1) is a unique spirooxindole alkaloid derived from a marine actinomycete and is characterized by its distinct spirocyclic pyrrolo[1,2-c]imidazolidin-4-one scaffold. Although we have successfully elucidated the biosynthetic pathway of 1, the mechanism underlying the formation of the characteristic imidazolidin-4-one remains unclear. In this study, we demonstrate that the cytochrome P450 monooxygenase CyaI catalyzes an oxidation reaction through a zwitterionic intermediate and facilitates a subsequent unusual C→N acetyl migration, which triggers a spontaneous intramolecular cyclization to forge the imidazolidin-4-one ring during 1 biosynthesis. In addition, CyaI is identified as a bifunctional enzyme that also catalyzes N-demethylation. High-resolution crystallography and mutagenesis studies determine Thr245 as a crucial catalytic residue that modulates the balance between imidazolidine-4-one synthesis and demethylation. This work not only expands the catalytic repertoire of P450 enzymes but also opens the way for the development of multifunctional biocatalysts in the synthesis of complex natural products.
S-adenosyl-L-methionine (SAM), an essential cofactor in all forms of life, is synthesized by the enzyme methionine adenosyltransferase (MAT) from methionine and ATP. The adenine moiety in SAM appears to have no direct function in catalysis, and some MAT homologs can utilize natural nucleotide triphosphates in vitro, producing the corresponding SAM nucleobase analogues. However, the molecular determinants of nucleotide choice of the MAT enzyme and the cellular significance of the nucleobase in SAM are unclear. In this study, using structure- and bioinformatics-guided mutagenesis, we identify a flexible active-site loop as a major determinant of nucleotide specificity in MAT. Loop mutations and loop swaps convert ATP-selective Escherichia coli MAT into variants that accept GTP, CTP, and UTP, enabling enzymatic synthesis and purification of S-guanosyl-, S-cytosyl-, and S-uracyl-L-methionine. Further, we show that these analogues partially rescue the growth of an E. coli SAM auxotroph under SAM-limited growth conditions. Biochemical assays show that the analogues bind the tested SAM-utilizing enzymes; they serve as substrates for E. coli SAM decarboxylase but do not support detectable methyl transfer by E. coli DNA adenine methyltransferase. These results establish the flexible loop as a gatekeeper of MAT nucleotide specificity and show that this loop can be engineered to produce SAM analogues which can selectively participate in downstream cellular metabolism. Graphical Abstract/ Table of contents only
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.
Chi-Fang Lee, T. Zhou, Songyi Xue et al.· Journal of the American Chem...· 0 citations
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced by biosynthetic enzymes that modify genetically encoded precursor peptide backbones and side chains. Genome mining and bioinformatics analyses targeting the multinuclear nonheme iron oxidative (MNIO) enzyme family led to the identification of a RiPP biosynthetic gene cluster from Streptomyces thermodiastaticus JCM 4840, the std cluster, which includes multiple biosynthetic enzymes and a precursor peptide containing a conserved SNKEWQE motif. Using in vitro approaches, we elucidated the modifications installed by the std biosynthetic enzymes. First, a YcaO-TfuA pair thioamidates the asparagine backbone. Next, a peptidase with an S8/S53 domain fused to a NodU-like carbamoyltransferase both carbamoylates the ε-amino group of lysine to produce the non-proteinogenic amino acid homocitrulline and cleaves the C-terminal EWQE motif. Finally, a partner protein-MNIO pair bis-hydroxylates the β- and γ-carbon positions of the installed homocitrulline to create dihydroxyhomocitrulline. The formation of homocitrulline and dihydroxyhomocitrulline is unprecedented in RiPP biosynthesis. Moreover, these findings expand the known substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyltransferases in these pathways.
Dayna P. Hebron, Tucker J. Shriver, Joshua J. Ziarek et al.· Journal of the American Chem...· 0 citations
Over the past several decades, biocatalysis has become a valuable complement to synthetic chemistry due to its high efficiency, exceptional selectivity, and environmental compatibility. Thiamine diphosphate (ThDP), the biologically active form of vitamin B1, serves as an essential coenzyme for core metabolic processes in all organisms. This review systematically elucidates the structural characteristics, classification, and diverse reactions catalyzed by ThDP-dependent enzymes, with a primary focus on their potential for stereoselective C-C bond formation and cleavage. These enzymes are widely distributed across all domains of life and catalyze the formation and cleavage of C-C, C-N, C-S, and C-O bonds. The catalytic mechanism centers on the formation of the Breslow intermediate, which undergoes nucleophilic addition to various electrophiles. Despite considerable sequence diversity, all ThDP-dependent enzymes share two conserved domains-the pyrimidine (PYR) binding domain and the pyrophosphate (PP) binding domain-and are classified into five structural types and nine superfamilies. In terms of substrate scope, the decarboxylase family is predominantly R-selective, whereas the transketolase family is S-selective. These enzymes hold significant promise for biotechnological applications, particularly through protein engineering to tailor catalytic activity and stereoselectivity. Moreover, ThDP-dependent enzymes have been implicated in the pathogenesis of Alzheimer's disease, diabetes, and tumor proliferation. This review also summarizes relevant clinical applications and the use of competitive inhibitors. By integrating modular architectures, cofactor synergy mechanisms, regulatory networks, and emerging frontiers in photoelectrochemical biocatalysis, this review highlights the broad potential of ThDP-dependent enzymes in both fundamental research and translational applications.
Jinxi Huang, Letong Huang, Xuemei Wang et al.· Organic and biomolecular che...· 0 citations
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.
Fang Pang, Yu-Ju Peng, Srinivas Thadkapally et al.· ACS Chemical Biology· 0 citations
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