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bioRxiv · 10.1101/2023.10.09.561484

Engineered Active Zymogen of Microbial Transglutaminase

Ariyoshi, R.·Matsuzaki, T.·Sato, R.·Minamihata, K.·Hayashi, K.·Wakabayashi, R.·Goto, M.·Kamiya, N.
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Abstract

Microbial transglutaminase (MTG) has shown to be a powerful biocatalytic glue for site-specific crosslinking of a range of biomolecules and synthetic molecules, those handled with an MTG-reactive moiety. The preparation of active recombinant MTG requires the posttranslational proteolytic digestion of propeptide working as an intramolecular chaperon to assist the correct folding of MTG zymogen (MTGz) in the biosynthesis. Herein, we propose an engineered active zymogen of MTG (EzMTG) that is expressed as soluble form in the host E. coli cytosol and exhibits the cross-linking activity without limited proteolysis. Based on the 3D structure of MTGz and serendipitous findings, saturated mutagenesis of K10 or Y12 in propeptide domain leads to generate several active MTGz mutants. In particular, K10D/Y12G mutant exhibited the catalytic activity comparable with a mature form. However, the expression level was low possibly due to the reduction of chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. By contrast, soluble K10R/Y12A mutant is expressed in the cytosol of host E. coli and exhibited unique substrate-dependent reactivity toward peptidyl substrates. The quantitative analysis of the binding affinity of mutated propeptide to the active site suggested the trade-off relationship of EzMTGs between the binding affinity and the catalytic activity. Our proof-of-concept study provides insights into the design of a new biocatalyst by using the zymogen as a scaffold and will convey a potential route to the high-throughput screening of MTG mutants for bioconjugation applications.

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Ariyoshi, R., Matsuzaki, T., Sato, R., Minamihata, K., Hayashi, K., Wakabayashi, R., Goto, M., Kamiya, N.. 2023-10-09. Engineered Active Zymogen of Microbial Transglutaminase. https://doi.org/10.1101/2023.10.09.561484

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