In the dynamic landscape of enzymology, protein kinases have long been recognized for their pivotal role in cellular signaling and regulation, primarily through the phosphorylation of substrate molecules using adenosine 5′-triphosphate (ATP). These enzymes orchestrate a plethora of physiological processes by transferring a phosphate group, thereby modulating the activity, localization, or interaction of target proteins. However, nestled within this vast superfamily are intriguing members known as pseudokinases—proteins that bear structural resemblance to canonical kinases yet lack traditional catalytic activity. These enigmatic proteins have perplexed scientists, traditionally classified as inactive enzymes due to absent or severely diminished phosphorylation capability. Now, groundbreaking research is peeling back the layers of pseudokinase functionality, revealing surprising enzymatic capacities that extend far beyond classical paradigms.
Recent investigations focusing on specific ribosomally synthesized and post-translationally modified peptides (RiPPs) have illuminated unexpected catalytic activities of pseudokinases. These peptides, notably from thioamitide and lanthipeptide families, undergo intricate chemical transformations, resulting in complex cyclic structures essential for their bioactivity. Two pseudokinases, TvaE and SacE, integral to these biosynthetic pathways, have been identified as key players in catalyzing peptide cyclization via thioether bond formation. This discovery challenges the traditional notion of pseudokinases as mere scaffolds or regulators, positioning them instead as active catalysts capable of forming (ene)thioether crosslinks within peptide substrates.
The core of this discovery lies in the enzymatic formation of unsaturated 2-aminovinyl-cysteine residues, a hallmark modification within thioamitides. Detailed biochemical assays confirm that TvaE facilitates this cyclization with remarkable specificity and efficiency. Complementing this, SacE orchestrates the formation of saturated lanthionine linkages, further substantiating the versatility of pseudokinase enzymes. These findings not only expand the functional repertoire of pseudokinases but also hint at a conserved mechanistic underpinning that could be widespread among these proteins.
Through a blend of heterologous expression systems, co-crystallization experiments, and computational modeling, a comprehensive picture of this unprecedented enzymatic activity emerges. Structural data reveals that these pseudokinases retain the canonical protein kinase fold, emphasizing that this ancient framework can be repurposed to catalyze chemistry distinct from phosphorylation. Remarkably, the cyclization reaction proceeds via a Michael addition mechanism, a nucleophilic conjugate addition well-known in organic chemistry but rarely associated with protein kinase domains.
The mechanics of this catalysis are elegantly complex. Instead of relying solely on active site residues, these pseudokinases employ a substrate-assisted process characterized by a sandwich-like interaction between the enzyme and the peptide substrate. This unique mode of action stabilizes transition states and facilitates the thioether bond formation, shedding light on a sophisticated evolutionary adaptation of enzymatic function. Such structural and mechanistic insights not only enhance our understanding of pseudokinases but also underscore the plasticity embedded within protein folds.
Genome mining efforts propelled by these discoveries identify a broader clade of pseudokinases harboring this cyclase function, suggesting that the capacity for peptide cyclization via thioether crosslinking is neither an isolated nor a rare phenomenon. This opens exciting avenues for exploring natural product biosynthesis and engineering novel peptide therapeutics. The biochemical versatility demonstrated by TvaE and SacE challenges the existing dogma that enzymatic activity invariably hinges on canonical phosphorylation, pointing to a rich landscape of enzymatic functions sculpted through evolutionary innovation.
Isotope labeling and site-directed mutagenesis experiments further validate the mechanism, pinpointing critical residues that mediate substrate binding and catalysis. These studies reveal that subtle structural features within the pseudokinase fold underpin the ability to engage in Michael addition chemistry, distinct from the phosphate transfer reactions typical of their active kinase cousins. This molecular delineation fosters an expanded definition of enzyme active sites, accommodating diverse catalytic strategies beyond classical frameworks.
The implications of these findings ripple through fields ranging from natural product chemistry to synthetic biology and drug development. By harnessing pseudokinases capable of catalyzing peptide cyclizations, scientists could design novel cyclic peptides with enhanced stability, bioavailability, and target specificity. Moreover, understanding how nature co-opts ancestral folds for new biochemical roles paves the way for engineering enzymes with tailored functions, potentially revolutionizing peptide-based therapeutics and biomaterials.
This research also highlights the importance of revisiting and characterizing pseudokinases, many of which remain poorly understood or dismissed due to presumed inactivity. The ability of pseudokinases to drive complex chemical transformations elevates their status from biological curiosities to bona fide enzymatic entities with potentially significant physiological and biotechnological roles. Future studies are poised to unravel the extent of this enzymatic diversity and its evolutionary origins, enriching our comprehension of protein function and enzymatic innovation.
In sum, the revelation that pseudokinases like TvaE and SacE can catalyze peptide macrocyclization via thioether crosslinking not only broadens the functional landscape of the protein kinase superfamily but also inspires a reevaluation of enzyme classification paradigms. This intersection of structural biology, enzymology, and natural product biosynthesis exemplifies the power of interdisciplinary research to unveil nature’s hidden catalytic strategies, setting the stage for transformative advances in molecular engineering.
The protein kinase fold, once solely tied to phosphate transfer, now emerges as a versatile scaffold capable of supporting radically different chemistry. Pseudokinases, far from being mere evolutionary relics, embody molecular innovation by facilitating cyclization reactions critical for producing biologically active cyclic peptides. These insights underscore the evolutionary creativity harnessed within protein domains, enabling organisms to generate chemical complexity through minimal structural rearrangements.
As research proceeds, the exploration of additional pseudokinases with uncharacterized activities will undoubtedly uncover further biochemical novelties. The methodological approach leveraging biochemical characterization, crystallography, computational simulations, and genome mining exemplifies a roadmap for future enzyme discovery and functional annotation. Collectively, these efforts enrich our molecular lexicon and expand the toolkit for bioactive compound synthesis.
Ultimately, this pioneering work refines our understanding of how protein folds can be repurposed, reveals untapped enzymatic potential within pseudokinases, and offers promising new strategies for synthetic biology and drug discovery. The convergence of enzymology with natural product chemistry thus continues to unveil nature’s ingenuity, reshaping the frontiers of molecular biology and biotechnology.
Subject of Research:
Function and catalytic mechanisms of pseudokinases in peptide cyclization and thioether crosslinking.
Article Title:
Pseudokinases can catalyse peptide cyclization through thioether crosslinking.
Article References:
Hu, L., Li, M., Sang, Y. et al. Pseudokinases can catalyse peptide cyclization through thioether crosslinking. Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01954-1
Image Credits:
AI Generated
