Evolutionary plasticity of regulatory--catalytic coupling in mono- and poly-ADP-ribosyl transferases
The evolution of modular proteins enables biological systems to diversify regulatory functions while preserving conserved catalytic activities. ADP-ribosyltransferases (ARTs), including mono-ADP-ribosyltransferases (MARylating enzymes) and poly-ADP-ribosyltransferases (PARylating enzymes), provide an ideal model for investigating how regulatory and catalytic domains co-evolve. Although ART catalytic domains are highly conserved, their regulatory architectures vary extensively, raising the question of whether regulatory domains determine catalytic activation, allosteric communication, and enzymatic output. Here, we systematically examined regulatory--catalytic interoperability by reconstituting evolutionarily distant ARTs from mammals, plants, and bacteria. We show that the regulatory domain of the MARylating enzyme human PARP3 (hPARP3) functionally complements the catalytic domains from the PARylating enzymes human PARP1 (hPARP1), human PARP2 (hPARP2), and Arabidopsis PARP2 (atPARP2), restoring robust DNA-break-dependent allosteric coupling for catalytic activation in response to both phosphorylated double-strand and single-strand DNA breaks. Complementation stimulates NAD+ consumption, promotes cis- and trans-poly(ADP-ribosyl)ation, and is accompanied by direct physical interactions between heterologous regulatory and catalytic domains. Unexpectedly, the strongest activation was observed between the hPARP3 regulatory domain and the atPARP2 catalytic domain, demonstrating conservation of productive allosteric communication across both enzymatic classes and species boundaries. In contrast, regulatory domains from PARylating ARTs failed to activate the catalytic domain of MARylating hPARP3, revealing a striking asymmetry in regulatory--catalytic compatibility. These findings identify conserved and specialised modes of allosteric communication within the ART superfamily and establish the concepts of general and specific combining ability as a framework for understanding modular protein evolution. More broadly, our results demonstrate that conserved regulatory--catalytic signalling networks can be functionally transferred between divergent enzymes, providing a foundation for mapping allosteric communication across ARTs and for engineering synthetic ADP-ribosylation systems with programmable regulatory inputs and catalytic outputs.