Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions.

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19F-labeling readily provided NMR detection of protein-drug and protein-protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19F-labeling method was 3.5-fold more sensitive than 15N-labeling, and could be combined with other chemical modification techniques such as lysine 13C-methylation. 13C-dimethylated-19F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

A two-step refolding of acid-denatured microbial transglutaminase escaping from the aggregation-prone intermediate.

Microbial transglutaminase (MTG) is a monomeric globular enzyme made of 331 amino acid residues. The conformation of MTG was examined over the pH 2.0-6.0 region using circular dichroism (CD) and 1-anilino-8-naphthalenesulfonate (ANS). Under conditions of low ionic strength, a decrease of pH below 4 caused a stepwise unfolding with an intermediate exhibiting specific ANS-binding before full unfolding at pH 2.0. At high ionic strength, the decrease of pH led to only an intermediate without further unfolding. The intermediate corresponds to the molten globule state with a secondary structure similar to the native state but disordered tertiary structures. A pH- and NaCl concentration-dependent phase diagram showed that the fully unfolded state exists only under limited conditions of low pH and a low NaCl concentration. Although a refolding yield by the direct jump to pH 6.0 was low, a two-step refolding with incubation at pH 4.0, where MTG is marginally stable, and a subsequent jump to pH 6.0 improved the yield by suppressing the kinetic traps. We propose that the two-step refolding is useful for improving the yield of larger proteins with a high pI value.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis.

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca(2+)-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase.

We previously observed secretion of native-type Streptomyces mobaraensis transglutaminase (MTGase) in Corynebacterium glutamicum by co-expressing the subtilisin-like protease SAM-P45 from S. albogriseolus which processes the pro-region. In the present study, we have used a chimeric pro-region consisting of S. mobaraensis and Streptomyces cinnamoneus transglutaminases for the production of MTGase in C. glutamicum. As a result, secretion of MTGase using the chimeric pro-region is increased compared to that using the native pro-region.

Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum.

We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain. As a result, native-type transglutaminase was secreted.

Structure of folding intermediates at pH 4.0 and native state of microbial transglutaminase.

Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.0 to 6.0. CD analysis showed that a burst of secondary structure formation occurred within the dead time of the experiment and accounted for 75% of the signal change in the far UV CD, with little tertiary structure being formed. This burst was followed by slow rearrangement of the secondary structure accompanied by formation of tertiary structure. The secondary and tertiary structures of the final sample at pH 4.0, corresponding to the folding intermediate, were different from these structures at pH 6.0. Once the native structure was obtained, acidification of the native protein to pH 4.0 did not lead to a structure like that of the folding intermediate. Sedimentation velocity analysis showed that the folding intermediate had an expanded structure and contained no other structure species including large aggregates.

Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus.

The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C. glutamicum but had no enzymatic activity. However, when the plasmid carrying the S. mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that C. glutamicum has potential as a host for industrial-scale protein production.

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.

Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense.

The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 A resolution. The protein folds into a plate-like shape, and has one deep cleft at the edge of the molecule. Its overall structure is completely different from that of the factor XIII-like transglutaminase, which possesses a cysteine protease-like catalytic triad. The catalytic residue, Cys(64), exists at the bottom of the cleft. Asp(255) resides at the position nearest to Cys(64) and is also adjacent to His(274). Interestingly, Cys(64), Asp(255), and His(274) superimpose well on the catalytic triad "Cys-His-Asp" of the factor XIII-like transglutaminase, in this order. The secondary structure frameworks around these residues are also similar to each other. These results imply that both transglutaminases are related by convergent evolution; however, the microbial transglutaminase has developed a novel catalytic mechanism specialized for the cross-linking reaction. The structure accounts well for the catalytic mechanism, in which Asp(255) is considered to be enzymatically essential, as well as for the causes of the higher reaction rate, the broader substrate specificity, and the lower deamidation activity of this enzyme.

Enhancement of transglutaminase activity by NMR identification of its flexible residues affecting the active site.

Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins, such as tofu, boiled fish paste, and sausage. By using nuclear magnetic resonance, we have shown that the residues exhibiting relatively high flexibility in MTG are localized in the N-terminal region; however, the N-terminal region influences the microenvironment of the active site. These results suggest that the N-terminal region is not of primary importance for the global fold, but influences the substrate binding. Therefore, in order to increase the transglutaminase activity, the N-terminal residues were chosen as candidates for site-directed replacement and deletion. We obtained several mutants with higher activity, del1-2, del1-3, and S2R. We propose a strategy for enzyme engineering targeted toward flexible regions involved in the enzymatic activity. In addition, we also briefly describe how the number of glutamine residues in a substrate protein can be increased by mixing more than two kinds of TGases with different substrate specificities.

NMR-based screening method for transglutaminases: rapid analysis of their substrate specificities and reaction rates.

Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins such as tofu, boiled fish paste, and sausage. Other transglutaminases (TGases) are expected to be used in the same way, and also to extend the scope of industrial applications to materials, drugs, and so on. The TGases have great diversity, not only in amino acid sequence and size, but also in their substrate specificities and catalytic activities, and therefore, it is quite difficult to estimate their reactivity. We have developed an NMR-based method using the enzymatic labeling technique (ELT) for simultaneous analysis of the substrate specificities and reaction rates of TGases. It is quite useful for comparing the existing TGases and for screening new TGases or TGases variants. This method has shown that MTG is superior for industrial use because of its lower substrate specificity compared with those of guinea pig liver transglutaminase (GTG) and red sea bream liver transglutaminase (FTG). We have also found that an MTG variant lacking an N-terminal aspartic acid residue has higher activity than that of the native enzyme.

Enzymatic labeling of arbitrary proteins.

This paper describes an enzymatic labeling technique (ELT), using transglutaminases. On the basis of the ELT, isotopic nuclei are easily incorporated into the gamma-carboxyamide groups of glutamine residues in arbitrary proteins, without changing their chemical structures. We have also shown that, by using ELT, protein aggregation was easily checked for NMR studies and that it can be applicable for the screening of weakly bound ligands for proteins. Owing to the simple preparation of the isotope-labeled proteins, ELT should be useful for speeding up various structural and functional analyses of arbitrary proteins.