The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method.

Biological regulation has long relied on modifying genes, transcripts, or protein abundance, yet direct rewriting of protein sequence after translation remains largely inaccessible. Here, we introduce a programmable protein editing platform based on split intein-mediated recombination, enabling precise excision and replacement of defined segments within mature proteins in living cells. By covalently exchanging a target segment with a donor-encoded sequence supplied in trans, this approach functions as a protein-level analogue of recombination, operating independently of transcription or translation. Using ultrafast and orthogonal split inteins, we demonstrate efficient protein recombination in mammalian cells, enabling single-amino acid substitutions, domain replacement, functional switching, and reprogramming of subcellular localization. This work establishes post-translational protein recombination as a general strategy for sequence-level control of protein function, expanding the conceptual and practical scope of biological editing beyond nucleic acids.

This study reports on the direct electron transfer (DET) ability of the enzyme spermidine dehydrogenase (SpDH) and its use in a DET-type enzymatic sensor for detecting spermine. SpDH was found to exhibit internal electron transfer from its cofactor, flavin adenine dinucleotide (FAD), to heme b. This was confirmed by observing the heme b-derived reduction peak at 560 nm in the presence of spermine, the substrate. SpDH was immobilized on a gold electrode via a dithiobis (succinimidyl hexanoate) self-assembled monolayer. The cyclic voltammetry analysis of the SpDH-immobilized gold electrode revealed an increased oxidation current in the presence of 0.1 mM spermine with an onset potential of −0.14 V vs. Ag/AgCl in the absence of an additional external electron acceptor. This result confirmed that SpDH is capable of DET. Chronoamperometric analyses were conducted using an SpDH-immobilized gold electrode with spermine as the substrate under a 0 V oxidation potential vs. Ag/AgCl using an artificial saliva matrix containing 10 µM ascorbic acid and 100 µM uric acid. The sensor exhibited good linear correlation between the current increase and spermine concentration from 0.2 to 2.0 µM, with a limit of detection of 0.084 µM, which encompasses the physiologically relevant spermine concentration found in the saliva. Primary structure alignments and 3D structure predictions revealed that all SpDH homologs possess two conserved histidine residues in the same location on the surface as the heme b ligand of SpDH. This indicates their potential for DET-ability with an electrode.

Nicotinoproteins are a group of NAD⁺-dependent dehydrogenases that bind NAD⁺ tightly and catalyze reactions without using free NAD⁺. In this study, we investigated the role of the unique insertion loop in nicotinoproteins. Carveol dehydrogenase (CADh), a short-chain dehydrogenase/reductase (SDR) nicotinoprotein, and β-hydroxybutyrate dehydrogenase from Alcaligenes faecalis (AfBHBDh), a non-nicotinoprotein counterpart, were used as model enzymes. An insertion loop-deleted mutant, CADh Δ39–49, was constructed. An insertion loop from Mycobacterium paratuberculosis CADh (MpCADh) was introduced into AfBHBDh to generate the two mutants. The results showed that CADh Δ39–49 lost NAD⁺ tight binding capacity and could not utilize free NAD⁺. In contrast, the AfBHBDh mutants showed no dye-mediated dehydrogenase activity. Moreover, the K_M and K_D values for NAD⁺ were higher than those of the wild-type enzyme. Docking simulations revealed a stronger binding affinity between NAD⁺ and the mutants than with the wild-type AfBHBDh. Taken together, these results suggest that the insertion loop interferes with NAD⁺ entry into the active site of the enzyme while creating a more energetically favorable binding environment. This loop is necessary but alone is insufficient to achieve NAD⁺ tight binding. This study deepens understanding of NAD⁺ binding in SDR nicotinoproteins and provides insights for SDR enzyme engineering.