Approaching transglutaminase from Streptomyces bacteria over three decades

Abstract

Transglutaminases are protein cross-linking and protein modifying enzymes that have attracted considerable interest due to their causal involvement in various diseases and versatility in industrial applications. In particular, microbial transglutaminases (MTG) from Streptomyces bacteria have managed in recent years to evolve from simple food additives to specialized enzymes for the sitedirected modification of therapeutic proteins. The review summarizes relevant studies from the beginning dealing with the occurrence, production, structure, catalysis, and substrate molecules of MTG enzymes. It also addresses biotechnological procedures with MTG from S. mobaraensis (SmMTG) as the most prominent representative in focus. Reassessment of the available data revealed unexpected insights into catalysis of SmMTG and other transglutaminases, suggesting selection of glutamine donor proteins by subsites at the front vestibule and the existence of distinct lysine pockets.Flexibility of the SmMTG-accessible glutamine donor substrate regions seems to be more important than the glutamine environment. Nevertheless, residues in close vicinity to glutamines also determine interaction with the SmMTG subsites. The apparent lack of subsites for lysine donor proteins suggests self-assembly of the substrate proteins prior to enzymatic cross-linking. The study of natural substrate proteins, especially their mutual interaction, is proposed to further illuminate catalysis of SmMTG. To this end, structure and function of the characterized substrate proteins from S. mobaraensis are discussed in conclusion.

Molecular crowding elicits the acceleration of enzymatic crosslinking of macromolecular substrates

“…The series of experiments described above reveal that protein–protein conjugation by MTG can be accelerated under crowded conditions. Although the biological role of MTG in the host organism is unclear, 9 the obtained results imply that the action of TGase with regard to macromolecular substrates may be intrinsically enhanced in a crowded environment, facilitating the formation of biologically important crosslinked products in nature. 37…”

“…8 Therefore, three factors affect enzyme activity under crowded conditions: the size of the crowding reagent, the volume excluded by the crowding reagent, and the size of the enzyme. Because enzymes have a wide substrate scope, 9 the molecular weight of a substrate should also affect the catalytic efficiency of the enzyme under crowded conditions. However, the effect of substrate size on an enzymatic reaction remains unclear because it is difficult to prepare a set of specific substrates with various molecular weights for an enzyme of interest.…”

Improved Productivity of Streptomyces mobaraensis Transglutaminase by Regulating Zymogen Activation

“…These results confirmed that NH 4 + increased the TAP activity at the early stage of fermentation. TAMP (purified from surface colonies on plates) was considered to be involved in TGase activation and regulated by SSTI in S. mobaraensis ( Zotzel et al, 2003a ; Juettner et al, 2018 ; Fuchsbauer, 2021 ). However, the transcript levels of TAMP and SSTI at 24 h were not changed in the presence and absence of NH 4 + (data not shown).…”

“…When treated at room temperature for 40 h, the TGase activity of the culture supernatant from the 48-h culture broth with NH 4 + addition retained 82% of initial activity, while that from 84-h culture broth without NH 4 + only obtained 65% residual activity ( Figure 3D ). This is probably due to the fact that a lot of proteases were produced at the later stage of S. mobaraensis , resulting in proteolytic degradation of TGase ( Fuchsbauer, 2021 ). Therefore, the reduced fermentation period could not only increase the economy of the TGase but also its storage stability.…”

Tag-free, specific conjugation of glycosylated IgG1 antibodies using microbial transglutaminase

“…Although mTG is widely used in biotechnology, the mechanism explaining its reactivity toward specific glutamine residues relative to others is not well understood. 14 Of particular interest, despite IgG1 human crystallizable fragment (hFc) antibody displaying 8 surface-exposed glutamines, 15 none shows reactivity towards mTG in the context of the native, glycosylated antibody ( Fig. 1A ).…”