In the dynamic world of protein chemistry, the quest to replicate the intricate biochemical processes that underpin life has always presented profound challenges. Among these is the mimicry of peptide bond formation—nature’s primary strategy for synthesizing proteins—where adenosine triphosphate (ATP) serves as a vital energy currency. In the most recent breakthrough, a team of researchers has elegantly harnessed this biological logic, developing an innovative ATP-driven platform that fundamentally transforms how scientists can activate and modify protein C termini. This work, grounded in the properties of a bacterial enzyme family related to ubiquitin-activating enzymes, signals a new era where high-yield peptide ligation becomes possible outside the confines of living cells.
Fundamentally, ATP is central to many biological processes, particularly those requiring energy-intensive transformations such as peptide bond formation. In natural biosynthesis pathways, ATP fuels the activation of carboxyl groups at peptide C termini, facilitating nucleophilic attack by amino groups of incoming amino acids or peptides, culminating in peptide bond formation. However, until now, translating this ATP-dependent activation to the laboratory bench has been a significant obstacle for protein chemists. The new study breaks this barrier by leveraging a bacterial enzyme known as MccB, an ancestral relative of the E1 ubiquitin-activating enzymes, to mimic ATP’s role in peptide bond chemistry—enabling controlled, ATP-fueled modifications of peptide and protein termini.
MccB belongs to a fascinating family of enzymes that have evolved to activate peptides through adenylation, a process where ATP is consumed to generate a highly reactive intermediate, often an adenylated ester. What the researchers found particularly compelling is MccB’s ability to form an O-AMPylated intermediate at the peptide C terminus—an electronegative species primed for nucleophilic attack. By engaging this O-AMP intermediate, the enzyme allows the introduction of a variety of nucleophiles that can modify the C-terminal end with diverse functional groups. Among these, thioesters stand out as especially versatile intermediates in both natural and synthetic protein chemistry, notably for applications in protein ligation and conjugation.
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Intriguingly, beyond merely activating peptides, the researchers tapped into the natural evolutionary diversity within the MccB enzyme family. By analyzing and mining these enzymes, they distinguished two functional classes: epitope-specific MccBs, which selectively target particular peptide sequences or proteins, and promiscuous MccBs, capable of modifying a broader spectrum of substrates. This duality opens remarkable possibilities in both targeted protein bioconjugation and broader synthetic applications. The targeted enzymes can be harnessed to direct modifications to specific proteins of interest, offering unprecedented precision, while the promiscuous enzymes provide a robust platform for generating reactive peptide thioesters useful in diverse downstream chemistries.
The ATP-driven strategy devised here mimics the elegant efficiency of biological peptide biosynthesis but operates under in vitro conditions, which is a significant leap forward. This inherently bio-inspired approach enables protein modifications with remarkable yield and specificity, sidestepping the limitations of traditional chemical peptide ligation techniques, which often require harsh reagents or non-physiological conditions. This synthetic versatility emphasizes the conceptual paradigm shift from merely understanding biological processes toward harnessing their mechanistic principles to engineer entirely new molecular tools.
From a mechanistic perspective, the chemistry underpinning this ATP-fueled activation is mechanistically resonant with canonical enzymatic adenylation reactions but demonstrates adaptability to non-native substrates—a feature that broadens the scope of chemical biology. The formation of the O-adenylated intermediate is biochemically analogous to the PHP or acyl-adenylate intermediates seen in natural peptide bond-forming enzymes. However, the engineered MccB platform extends this utility by mediating subsequent nucleophilic attacks by exogenous molecules, generating stable thioester or other functionalized moieties that are essential for efficient peptide ligation or protein functionalization.
The implications of this development are multifold. For protein engineering, this provides a powerful new toolkit for manipulating protein termini in a controlled manner under ATP-driven conditions, which faithfully recapitulate natural biochemical logic. Such tailored modifications can be pivotal for creating protein conjugates, labeling specific sites, or generating novel therapeutic modalities. For chemical synthesis, the ability to produce peptide thioesters enzymatically presents an elegant alternative to synthetic methods, which may struggle with yields or scope. Moreover, the possibility to direct MccB enzymes to distinctive epitopes within target proteins advocates a future where site-specific modifications become routine and scalable.
This work also brings into focus the evolutionary kinship shared between bacterial peptide-activating enzymes and eukaryotic ubiquitin activation systems. While E1 enzymes play crucial roles in post-translational tagging of proteins, repurposing their ancestral bacterial counterparts for synthetic ends exemplifies the power of directed enzyme engineering informed by evolutionary biology. By bridging this lineage with chemical innovation, the researchers have expanded the repertoire of enzymatic transformations that can be precisely controlled and harnessed in a lab setting.
Exploring the substrate specificity of these MccB variants revealed subtle yet significant differences in their active site architecture and dynamics, which dictate their promiscuity or selectivity. This insight facilitates future protein engineering efforts aimed at fine-tuning enzyme behavior for applications requiring either high specificity or broad-spectrum reactivity. The experimental data suggests that epitope-specific MccBs might be especially useful in complex biological contexts where off-target modification must be minimized.
Furthermore, the adoption of this platform for protein C-terminal bioconjugation—mediated by the generation of activated thioester intermediates—offers a streamlined path toward constructing well-defined protein conjugates. Such constructs are valuable for myriad applications, including drug delivery, imaging, and synthetic biology. Enzymatically generated thioesters retain the high reactivity and versatility observed in native protein ligation chemistry but with the advantage of preparation under mild, biologically compatible conditions, circumventing many of the challenges posed by chemical approaches.
The method also aligns with the broader trend in chemical biology of moving toward enzymatically guided synthesis and modification of biomolecules, which tend to maximize selectivity and efficiency while reducing environmental and procedural burdens. The reliance on ATP, a ubiquitous and biologically benign energy source, further underscores the sustainable appeal of this strategy. This ATP-driven enzymatic activation can potentially integrate seamlessly with other biotechnological workflows, ranging from in vitro protein engineering to cell-free synthetic biology systems.
Looking ahead, the versatility of the MccB platform primes it for further expansion. The capacity to adapt enzyme specificity through protein engineering, combined with the modularity of nucleophilic partners, hints at a future where peptide and protein functionalization becomes an exactly tunable, routine procedure. Such control could extend into therapeutics, biosensor development, and complex molecular assembly lines, revolutionizing the manipulation of protein structures and enabling discoveries previously hampered by limitations in synthetic accessibility.
In conclusion, this landmark study not only presents a novel enzymatic strategy for ATP-driven protein C-terminal modification but also exemplifies how a deep mechanistic understanding of enzyme evolution and function can yield transformative tools for chemistry and biology. By recreating the energetic logic of peptide bond biosynthesis in vitro, the researchers have crafted a precise, high-yielding platform for protein modification that transcends prior technical limitations. This development promises to invigorate research in protein bioconjugation, synthetic biology, and enzyme engineering, driving forward our capabilities to construct and manipulate proteins with exquisite molecular precision.
Subject of Research: Engineered enzymatic systems for ATP-driven activation and modification of peptide and protein C termini
Article Title: Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein and peptide C termini
Article References:
Frazier, C.L., Deb, D., Leiter, W.E. et al. Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein and peptide C termini. Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01871-3
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Tags: advances in synthetic biologyATP-driven protein modificationBacterial enzyme researchbiochemical processes in protein chemistryenergy currency in biologyhigh-yield peptide ligationinnovative protein modification techniqueslaboratory protein synthesis challengesnucleophilic attack in peptide chemistrypeptide bond formationprotein C-terminus activationubiquitin-activating enzymes