Artificial metalloenzymes based on the Spy system exhibit versatile catalytic functions, from catecholase activity to hydrogen evolution

Metalloproteins catalyze some of the most complex biochemical transformations in nature. This study explores the rational redesign of the Spy system, comprising a peptide (SpyTag) that spontaneously forms a covalent isopeptide bond with its protein partner (SpyCatcher), yielding a stable recombinant Spy protein. The modular architecture of the system offers distinct advantages for the development of artificial metalloenzymes: the peptide segment permits the strategic incorporation of metal-binding motifs without requiring full protein reengineering, while its compatibility with solid-phase peptide synthesis allows for the introduction of non-canonical amino acids. These features collectively expand the design space of Spy-based constructs, paving the way for novel catalytic systems with potential applications in biotechnology, nanotechnology, and industrial catalysis. We investigated second-generation SpyTag peptides engineered with metal-binding motifs (Figure1): bis-histidine sites for Cu(II) coordination, enabling catechol oxidation, and cysteine-based motifs for Ni(II) binding, targeting hydrogen evolution catalysis. To evaluate the oxidative activity toward catechols, 4-methylcatechol was used as a model substrate, while L- and D-DOPA were tested to assess enantioselectivity. A biphasic system was also explored to enable catalyst reuse by replacing only the consumed catecholic substrate. By exploiting the modular architecture of the system, we also insert a nickel-binding cysteinerich motif at the N-terminus of SpyTag and expressed SpyCatcher on the surface of Escherichia coli. Enabling rapid assembly of a construct capable of producing hydrogen.