Tomoko Ohtsuka

Crystal Structure of Red Sea Bream Transglutaminase

The crystal structure of the tissue-type transglutaminase from red sea bream liver (fish-derived transglutaminase, FTG) has been determined at 2.5-Å resolution using the molecular replacement method, based on the crystal structure of human blood coagulation factor XIII, which is a transglutaminase zymogen. The model contains 666 residues of a total of 695 residues, 382 water molecules, and 1 sulfate ion. FTG consists of four domains, and its overall and active site structures are similar to those of human factor XIII. However, significant structural differences are observed in both the acyl donor and acyl acceptor binding sites, which account for the difference in substrate preferences. The active site of the enzyme is inaccessible to the solvent, because the catalytic Cys-272 hydrogen-bonds to Tyr-515, which is thought to be displaced upon acyl donor binding to FTG. It is postulated that the binding of an inappropriate substrate to FTG would lead to inactivation of the enzyme because of the formation of a new disulfide bridge between Cys-272 and the adjacent Cys-333 immediately after the displacement of Tyr-515. Considering the mutational studies previously reported on the tissue-type transglutaminases, we propose that Cys-333 and Tyr-515 are important in strictly controlling the enzymatic activity of FTG.

Enhanced susceptibility to transglutaminase reaction of α-lactalbumin in the molten globule state

The susceptibility of alpha-lactalbumin to transglutaminase reactions was studied using an enzyme from Streptoverticillium which can catalyze the reactions irrespective of the presence or absence of Ca2+. Transglutaminase-catalyzed polymerization of alpha-lactalbumin in the native state occurred to a very limited extent. Transformation from the native state to the molten globule state brought about by Ca(2+)-removal from holo-alpha-lactalbumin enhanced the polymerization of the protein catalyzed by transglutaminase. The incorporation of Carbobenzoxy-Gln-Gly into alpha-lactalbumin through the enzyme reaction was investigated to determine the amounts of lysine residues which are present at molecular surface and available to the enzyme. There was no significant difference in the amount of available lysine residues between the native and the molten globule molecule. However, the amount of surface glutamine residues incorporated with monodansylcadaverine by transglutaminase was remarkably higher in the molten globule state than that in the native state. The monodansylcadaverine-incorporated site of alpha-lactalbumin in the molten globule state was identified as Gln-54 by amino-acid sequence analysis of fluorescence-labeled peptides separated from chymotryptic digests of the protein. Possible reason for selective labeling of Gln-54 in molten globule alpha-lactalbumin was proposed.

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence.

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.