Aug 2026· Angewandte Chemie· pp.
e7360177
· 0 citations· 58 references
Medicine
TL;DR
It is demonstrated 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.
Abstract
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.
The oxidative modification of complex natural products is critical for their biological activity. However, the enzymatic mechanisms that facilitate three-step, six-electron transfer reactions, such as the conversion of a methyl group to a carboxylic acid, on cyclopeptide scaffolds remain poorly understood. This study characterizes IlaL, a cytochrome P450 monooxygenase from Streptomyces atratus, which catalyzes a six‑electron methyl‑to‑carboxylic acid conversion during the biosynthesis of the potent antimycobacterial cyclopeptide ilamycins. IlaL possesses a noncanonical “Asp-Asn (DN)” dioxygen activation dyad and an extended, flexible BC-loop, which together enable sequential three-step oxidation. The high-resolution co-crystal structure of IlaL bound to the substrate ilamycin B2 reveals an enlarged cavity and dynamic gating conferring precise regioselectivity and broad substrate tolerance. Biochemical and phylogenetic analyses confirm the essential role of the DN dyad and show that DN‑type cytochrome P450 monooxygenases occupy a distinct, recently emerged phylogenetic clade. These findings provide structural and mechanistic insights into six‑electron oxidative modification and establish design principles for versatile biocatalysts enabling late‑stage natural product diversification.
During the biosynthesis of the peptide antibiotic rufomycin, the cytochrome P450 (P450) enzyme RufO catalyzes the aromatic nitration of a tyrosine residue in a ribosomal pentapeptide that serves as a precursor for nonribosomal peptide synthesis. To understand the mechanism of this unusual P450-mediated reaction, a series of pentapeptides were tested as substrates alongside molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM, ONIOM approach) calculations. A new substrate-bound crystal structure of the homologue NsRufO was also obtained. These experiments revealed the intimate and necessary involvement of a histidine residue within the pentapeptide substrate of RufO in supporting effective nitration, with the protonation state of the intermediate compound II proposed to influence the production of nitrated tyrosine over unwanted nitrate formation. These findings provide key insights into the mechanism of P450-mediated nitration that explain how sequence differences in RufO and its homologues, along with the structure of the pentapeptide substrate, facilitate aromatic nitration of tyrosine by RufO.
A. M. Kirk, Jemma Gullick, Yong-Wei Zhao et al.· Journal of the American Chem...· 0 citations
Natural product biosynthetic cytochrome P450s (CYPs) are of increasing interest for their incredible ability to selectively activate inert C–H bonds on highly complex substrates. However, these enzymes are understudied due to difficulties accessing substrates. Here, we use our recently developed peptide cyclisation chemistry to produce a library of natural product peptide analogues to study the rufomycin tailoring CYP, RufM. Rufomycins are antimicrobial non-ribosomal cyclic peptides that undergo sequential alkyl oxidation catalysed by RufM. This transformation is essential for rufomycin antimycobacterial activity but results in several naturally produced derivatives, of which one intermediate has the greatest activity. Due to current interest in engineering the rufomycin pathway to generate derivatives for improved properties, we use our peptide library to explore the complex interplay of enzyme catalysed and spontaneous transformations involved in RufM oxidation, suprising RufM substrate promiscuity and the role of a mechanistically important residue in sequential oxidation. Our work demonstrates the power of our chemistry both as a tool to study biosynthetic tailoring CYPs as potential late-stage functionalizing biocatalysts and to enable antibiotic development.
Jessica Peate, Yao-Yu Ding, Joshua Tomkins et al.· Chemical Science· 0 citations
Glaucocalyxin A (GLA), a well-known ent-kaurene diterpenoid, has emerged as a high-value bioactive natural product in pharmaceutical research owing to its potent and broad-spectrum activities. To date, its total synthesis has proven extremely challenging due to poor yield and intricate routes, and the biosynthetic pathway of GLA remains poorly understood. Herein, we identified two enzymes, IamCYP71D761 and IamCYP706V18, in Isodon amethystoides, which specifically mediate the C3α- and C15β-hydroxylation of ent-kaurene with high stereoselectivity and regioselectivity, respectively, generating 3α,15β-dihydroxy-ent-kaurene (3), a key precursor in the biosynthesis of GLA. In addition, we established an efficient de novo biosynthesis platform for 3 in Saccharomyces cerevisiae. Briefly, high-efficiency isozyme screening, protein engineering, increased acetyl-CoA synthesis, and copy number enhancement were applied to the ent-kaurene biosynthesis module. Subsequently, promoter optimization, competitive pathway knockout, and electron transfer optimization were introduced, resulting in production of 3 to 24.0 ± 0.2 mg/L in shake flask. In summary, this work highlights the pivotal roles of IamCYP71D761 and IamCYP706V18 in the heterologous biosynthesis of 3, offering valuable insights for the further pathway reconstruction and de novo production of GLA, GLB, and other ent-kaurene diterpenoids.
Yanying Chen, Xinqi Song, Ya-Nan Wang et al.· Synthetic and Systems Biotec...· 0 citations
Daphnetin is a clinically established coumarin, but its native biosynthetic pathway remains elusive. In this study, a 2-oxoglutarate-dependent dioxygenase (2OGD), AtS8H, was identified that hydroxylates umbelliferone (UMB) to daphnetin , enabling the design of an artificial biosynthetic pathway. However, the pathway efficiency was constrained by the poor solubility of AtS8H and the low catalytic activity of the upstream 2OGD enzyme IbC2'H. To enhance AtS8H solubility, we developed an integrated strategy combining ProteinMPNN-guided sequence redesign with surface charge engineering. The obtained quintuple mutant S8H 2-6 exhibited significantly improved solubility and a 7.2-fold increase in catalytic efficiency. For IbC2'H, we developed a fluorescence-based high-throughput screening method, and a quadruple mutant C6 was obtained by directed evolution, which displayed a 2.7-fold higher kcat and a 3.2 °C improvement in thermal stability. Implementing both engineered enzymes into an optimized Escherichia coli strain enabled the de novo production of daphnetin at a titer of 46 mg/L. This work reports, to the best of our knowledge, the first microbial de novo production of daphnetin from a simple carbon source and demonstrates an integrated enzyme engineering approach that synergistically refines biosynthetic pathways for efficient microbial production.
Shunmin Ji, Chong Xie, Yanyan Wang et al.· ACS Synthetic Biology· 0 citations
Bacterial azabicyclocarbamates and related pyrrolizidine alkaloids play important roles in microbial interactions, and are scaffolds of therapeutic potential. Their biosynthesis involves a bimodular non-ribosomal peptide synthetase (NRPS), as well as a Baeyer-Villiger monooxygenase and tailoring enzymes, the latter contributing to the structural diversification of these compounds. Azetidomonamide A, a core metabolite produced by the major human opportunistic pathogen Pseudomonas aeruginosa, is a rare 4,7-bicyclocarbamate involved in modulating bacterial virulence that belongs to a unique family of natural products targeting ClpP proteases. In this study, we elucidated the full set of reactions leading to the 7-membered cyclocarbamate warhead, and reconstituted in vitro the biosynthesis of azetidomonamide A. Notably, this approach allowed for detailed characterization of a condensation (C) domain-catalyzed online dehydration via chemical capture of NRPS-tethered intermediates. Furthermore, we identified the dehydratase AzeD as the founding member of a distinct group of standalone proteins of the C domain family. Via combined structural, docking and biochemical analyses, we provided evidence that AzeD’s catalytic mechanism is distinct from that of dehydrating C domains, further expanding the known chemistry of these key biosynthetic enzymes.
Xiaohan Liu, S. Najah, Andrea Calderari et al.· bioRxiv· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
