In a groundbreaking advance in ribosomally synthesized and posttranslationally modified peptides (RiPPs) biosynthesis, researchers have unveiled a revolutionary enzymatic mechanism that repeatedly and selectively modifies mature lasso peptides — a structural feat unprecedented in natural product biosynthesis. Until now, the scientific consensus held that iterative enzymatic modifications predominantly targeted linear precursors. The intricate three-dimensional scaffolds of mature RiPPs posed a formidable challenge, due in part to the complex dynamic changes required for substrate recognition and modification. This paradigm has now been upended by a prolific class of GCN5-related N-acetyltransferases (GNATs) that catalyze multiple, consecutive acylations on fully folded lasso peptide substrates, fundamentally broadening the biochemical repertoire and structural diversity achievable through natural enzymatic pathways.
Lasso peptides, distinguished by their unique knot-like topology with a ring-and-loop motif, are notable for their remarkable stability and bioactive properties. They have captivated researchers for decades, offering therapeutic potential and intriguing biochemical puzzles. The newly characterized GNATs add yet another layer of complexity to lasso peptide biosynthesis, as they specifically acetylate two lysine residues positioned within spatially distinct regions — the loop and ring — of mature peptides. This spatially resolved iterative acylation diverges clearly from the canonical single-step or linear poly-modification models, highlighting an elegant enzymatic strategy to expand chemical complexity and functional diversity in natural products.
Central to this discovery is the structural elucidation of the peptide-binding pocket in the GNAT enzyme IatT, achieved through state-of-the-art cryogenic-electron microscopy (cryo-EM). The cryo-EM data provides an unprecedented atomic-level depiction of how IatT recognizes and accommodates mature lasso peptides. Unlike typical enzymes that engage flexible, linear peptide chains, IatT displays conformational plasticity and a highly specialized binding site that precisely orients the mature lasso peptide for sequential lysine acetylation. Key residues within the binding pocket selectively mediate the first and second acetylation events, with subtle shifts in interactions guiding the enzyme’s function from one lysine site to the next.
This elegant structural choreography not only clarifies the molecular basis of iterative catalysis on a folded protein substrate, but it also reveals new paths for enzyme engineering. By rational design, the authors successfully expanded the substrate-binding cavity of IatT, enabling it to accept and transfer longer-chain acyl groups beyond acetyl moieties. This structural modification effectively converts IatT into a lipolasso peptide synthetase, generating ribosomal lipopeptides—a novel class of natural product with potentially enhanced membrane interactions and bioactivities. This breakthrough holds significant pharmaceutical promise, as lipopeptides frequently exhibit improved stability, target affinity, and permeability, properties highly coveted in drug development.
Beyond the confines of lasso peptides, this study illuminates a broadly applicable enzymatic strategy for diversifying RiPPs. GNATs are widely encoded within RiPP biosynthetic gene clusters, suggesting that iterative acylation could be a widespread natural mechanism and offering an expansive toolkit for synthetic biology and drug discovery. The potential to systematically reprogram GNATs to install various acyl chains on diverse peptides could revolutionize the production of novel bioactive compounds with tailored properties, heralding a new era of modular RiPP diversification.
The discovery challenges traditional views on substrate specificity in posttranslational modifying enzymes, particularly among acetyltransferases, which were conventionally thought to operate on linear, flexible peptides or unstructured substrates. The ability of IatT and related GNATs to robustly engage the highly structured lasso topology and catalyze distinct sequential modifications underscores an advanced evolutionary adaptation that reconciles enzyme flexibility with substrate complexity.
Mechanistically, this iterative modification involves finely tuned substrate recognition and product release steps that allow the enzyme to toggle between catalytic states without fully releasing the peptide intermediate, thereby increasing catalytic efficiency. This is especially remarkable given the conformational rigidity and entrapment inherent in mature lasso peptides. The enzymatic finesse demonstrated by these GNATs opens new investigative avenues into allosteric regulation, enzyme dynamics, and the evolutionary pressures that shaped these multifunctional enzymes.
Moreover, the structural insights gained from cryo-EM provide a blueprint for future engineering efforts. By pinpointing amino acid residues key to substrate binding and catalysis, researchers can now design GNAT variants with customized recognition profiles and catalytic capabilities. This modular approach to enzyme engineering could accelerate the biosynthesis of designer RiPPs featuring chemically diverse acyl decorations, expanding the chemical space accessible through biocatalysis and green chemistry.
The generated lipolasso peptides, formed by incorporating longer acyl chains via engineered IatT, represent an exciting new chemical class with potential utility as antimicrobial agents, biosurfactants, or signaling molecules. Their amphipathic nature, conferred by the lipid substitution, might grant them novel modes of action and biological targets, including improved membrane disruption or receptor binding, properties highly sought after in therapeutic agent development.
This study also establishes a framework for mining genomic databases to identify and characterize previously unrecognized GNATs capable of iterative modification. Equipped with structural and functional signatures from IatT, bioinformatic approaches can predict new enzyme candidates for experimental validation, thereby catalyzing a new wave of natural product discovery that leverages enzyme multifunctionality and structural plasticity.
Importantly, the findings have broad implications beyond RiPPs, as GNATs and acetyltransferases fulfill critical roles in diverse biological contexts ranging from histone modification to antibacterial resistance. Understanding how these enzymes achieve iterative modifications on complex substrates could inform the design of novel inhibitors or chemical probes targeting analogous mechanisms in pathogenic organisms or disease states.
From a synthetic biology standpoint, this work offers a strategic template for assembling hybrid biosynthetic pathways that incorporate engineered GNATs, thereby expanding the molecular diversity and functional tuning of natural product libraries. The ability to integrate iterative acylation steps on fully mature substrates positions these enzymes as versatile biocatalysts amenable to high-throughput screening and combinatorial biosynthesis platforms.
Ultimately, this discovery melds structural biology, enzymology, and chemical engineering to reshape our understanding of posttranslational modification dynamics and offers tangible strategies for harnessing enzymatic multifunctionality. By revealing the capacity of GNAT acetyltransferases to iteratively functionalize complex peptide architectures, the work lays the groundwork for novel classes of bioactive compounds and enriches the molecular toolbox of natural product chemistry.
As the search continues for novel antimicrobial agents and peptide-based therapeutics, this newfound enzymatic versatility underscores the untapped potential residing within RiPP biosynthetic pathways. The prospect of efficiently diversifying peptide natural products through iterative acylation not only accelerates drug discovery but also enriches our molecular understanding of enzyme-substrate co-evolution in nature’s chemical arsenal.
This pioneering research exemplifies how cutting-edge structural and biochemical techniques can converge to unlock new biological paradigms while providing inspiring avenues for translational innovation. The success in redefining enzymatic iterative catalysis to mature lasso peptides signals a transformative shift—a powerful testament to the evolving complexity and sophistication within natural product biosynthesis.
Subject of Research: Iterative enzymatic acylation of mature lasso peptides by GCN5-related acetyltransferases (GNATs) and engineering of these enzymes to expand RiPP structural diversity.
Article Title: Iterative acylation on mature lasso peptides by widespread acetyltransferases.
Article References:
Xiong, J., Wu, S., Liang, ZQ. et al. Iterative acylation on mature lasso peptides by widespread acetyltransferases. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-026-02149-6
Image Credits: AI Generated
