Jul 2026· Journal of the American Chemical Society· Vol 148, pp. 32426 - 32439· 0 citations· 55 references
Medicine
TL;DR
In vitro approaches elucidated the modifications installed by the std biosynthetic enzymes and expand the known substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyltransferases in these pathways.
Abstract
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.
Nonheme iron/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) catalyze diverse oxidative transformations, including hydroxylation, desaturation, and ring expansion. Although these reactions are well established, Fe/αKGD-catalyzed epimerization remains rare and has not been characterized in ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report the RiPP-modifying enzyme YmI, a bifunctional Fe/αKGD that catalyzes Cβ-hydroxylation of Phe19 and Cα-epimerization of Ile20 on the precursor peptide YmA toward the formation of YM-216391. The hydroxylation generates (2S,3R)-β-hydroxyphenylalanine, whose l-threonine-like stereochemistry facilitates subsequent formation of an unusual 5-phenyloxazole moiety. Substrate profiling analyses reveal leader peptide dependence for catalytic activity. Structural and docking studies provide insights into the substrate binding and active site architecture. Deuterium labeling demonstrates that YmI catalyzes Ile epimerization by directly abstracting the Cα-hydrogen, generating a Cα radical that is subsequently reprotonated by a nonexchangeable hydrogen source within the enzyme. These results unveil an unprecedented mechanism and novel bifunctionality, markedly expanding the reaction landscape for αKG-dependent enzymes in RiPP biosynthesis.
Unknown authors· Journal of the American Chem...· 0 citations
Condensation (C) domains in nonribosomal peptide synthetase (NRPS) pathways exhibit versatile functions that drive biosynthetic and chemical novelty. Through genome mining for atypical C domains, we identified a hybrid NRPS/polyketide synthase (PKS) biosynthetic gene cluster (mxg) from Cystobacterineae sp. MCy9003 and discovered myxoglucamides, a family of glycolipopeptides featuring an unprecedented vinyl-substituted γ-amino acid bearing an α-hydroxy/α-ketoamide functionality. Heterologous expression of the promoter-refactored pathway revealed new O-acylated myxoglucamides, and subsequent studies unveiled the C domain-like enzyme MxgH as a promiscuous O-acyltransferase decorating the glucose moiety with short-chain acyl groups. Biosynthetic investigations demonstrated that the unusual γ-amino acid originates from l-glutamate. Completion of the cryptic β-hydroxylation of peptidyl carrier protein-tethered glutamate by the α-ketoglutarate-dependent dioxygenase OxMxgA occurs only concomitantly with upstream chain extension, revealing a bidirectional checkpoint for substrate fidelity. Unexpectedly, the C-domain-like interface domain IMxgB is dispensable for this coupled transformation. Mutational analysis of the FMN-dependent monooxygenase encoded by mxgE, together with characterization of a shunt metabolite, supported its role in α-oxidation for α-hydroxy/α-ketoamide formation during γ-amino acid assembly. Together, these findings uncover an unrecognized biosynthetic logic for generating vinyl-substituted, α-oxidized γ-amino acids and substantially expand the functional repertoire of NRPS/PKS assembly lines.
Tingting Wang, Alexander Popoff, Maja Hunter et al.· Angewandte Chemie· 2 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
Canonical terpenoids conform to the (C5H8)n formula, whereas an emerging group of terpenoids that break the isoprene rule have been discovered recently. The precursor of C17 homosesquiterpenoid chlororaphens is derived from converting canonical C15 farnesyl pyrophosphate (FPP) by two consecutive methyltransferases (MTs). The first step is catalyzed by a bi-functional S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates C10 on FPP to trigger a cyclization cascade and generate the monocyclic C16 γ-presodorifen pyrophosphate (γ-PSPP). Here, we report the crystal structures of γ-PSPP synthase from Variovorax boronicumulans PHE5-4 (VbFPPMT) and Pseudomonas chlororaphis O6 (PcFPPMT), as well as the Mg2+ ion- and geranyl pyrophosphate (GPP) -bound complex of PcFPPMT. These structures reveal the overall structure of γ-PSPP synthases, composition of the catalytic center, and a flexible region responsible for the anchoring of the pyrophosphate (PPi) moiety of the substrate and a Mg2+ ion. Combining structural analysis, molecular docking, and site-directed mutagenesis, we identified key residues that govern the catalytic mechanism of these bi-functional γ-PSPP synthases. These findings advance our understanding into the catalytic mechanism employed by non-canonical methyltransferases, establish a vital framework for further mechanistic understanding and benefit rational engineering of C16/C17 terpenoid biosynthesis.
Xiuqin Li, Jian-Wen Huang, Lilan Zhang et al.· International Journal of Bio...· 0 citations
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.
Zhongtian Yu, Wenhe Zhang, Richiro Ushimaru et al.· Journal of the American Chem...· 0 citations
The multinuclear nonheme iron-dependent oxidative enzyme (MNIO) family employs a multi-iron cofactor to catalyze a range of post-translational modifications (PTMs) in the biosynthesis of ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. While significant progress has been made toward understanding the range of chemical transformations performed by MNIOs, the nature of the iron cofactor has only been investigated in one instance. Here, we examine the MNIO involved in oxazolin biosynthesis to gain further insight into the metallocofactors employed by this impressive family of enzymes. Oxazolin, a RiPP virulence factor from nontypeable Haemophilus influenzae, contains six copper-binding 5-thiooxazole groups installed by the MNIO HvfB. Weak interactions between HvfB and its required partner protein, HvfC, motivated genetic fusion of the two proteins, which yielded an effective mimic of the protein complex with high enzymatic activity. While HvfB binds up to three iron ions, concerted EPR, ENDOR, and Mössbauer spectroscopic characterization of the active protein reveals that accumulation of a mixed-valent diiron(II/III) cluster correlates with 5-thiooxazole product formation. This oxidation state is attained only in the presence of HvfC, revealing a new role for the partner protein in modulating the iron cofactor. Site-directed mutagenesis of metal-coordinating residues was used to probe the function of the third iron-binding site. This work clarifies the nature of the active iron cofactor for oxazolin maturation, providing a second example of a mixed-valent diiron oxidase in RiPP biosynthesis. TOC Graphic
Olivia M. Manley, Madeline B. Ho, Patrick M. McLean et al.· bioRxiv· 0 citations
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