Crystal Structure of Microbial Transglutaminase fromStreptoverticillium mobaraense

Abstract

The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 Å resolution. The protein folds into a platelike 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. superimpose well on the catalytic triad "Cys-HisAsp" 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.Transglutaminase (TGase 1 ; protein-glutamine ␥-glutamyltransferase, EC 2.3.2.13) catalyzes an acyl transfer reaction in which the ␥-carboxyamide groups of peptide-bound glutamine residues act as the acyl donors. The most common acyl acceptors of TGase are the ⑀-amino groups of lysine residues within peptides or the primary amino groups of some naturally occurring polyamines (1, 2). When lysine residues in proteins serve as acyl acceptors, intermolecular or intramolecular ⑀-(␥-glutamyl)lysine bonds are formed, resulting in the polymerization of proteins.TGases are widely distributed in various organisms, including vertebrates (3-7), invertebrates (8, 9), mollusks (10), plants (11), and microorganisms (12). Among these TGases, the human blood coagulation factor XIII has been most characterized (13)(14)(15)(16)(17)(18). By catalyzing the cross-linking between fibrin molecules, factor XIII forms fibrin clots for hemostasis and heals a wound. The crystal structure of human factor XIII has been determined, revealing that it consists of four domains with a cysteine protease-like active site (19 -22). Many TGases are homologous to human factor XIII and share the common feature of Ca 2ϩ -dependent catalytic activity (3-8). A tissue-type TGase from red sea bream liver (fish-derived TGase (FTG)) is an example of such factor XIII-like TGases and shows 33% sequence homology to human factor XIII (7). The crystal structure of FTG has also been determined (23). The overall and active site structures of FTG are essentially similar to those of human factor XIII.A microbial TGase (MTG) has been isolated from the culture medium of Streptoverticillium sp. S-8112 (24), which has been identified as a variant of Sv. mobaraense. This enzyme is the first TGase obtained from a nonmammalian source. Thus far, few TGases have been identified from microorganisms, particularly from Streptoverticillium species (25). Although the physiological role of MTG is still unknown, this protein is secreted from the cytoplas...

Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation

“…From the type of chemical reaction, transglutaminases, which replace the NH 2 -group of the amide of glutamine by another amine residue, are closely related to deamidases, which change the amide to a carboxylate. Transglutaminases possess a similar catalytic triad as PMT and papain proteases (33)(34)(35). For example, the catalytic triad (Cys-His-Asp) of the transglutaminases factor XIII perfectly matches the position of the catalytic triad of PMT (Fig.…”

“…To this end, G␤␥ was transcribed and translated in vitro in the presence of [ 35 S]methionine. The resulting 35 Slabeled G␤␥ was pulled down with GST-tagged G␣ i2 , which was purified from coexpression with either active or inactive PMT-C. G␣ i2 obtained from coexpression with active PMT-C wt exhibited diminished G␤␥-binding in the presence of GDP compared with G␣ i2 obtained from coexpression with inactive PMT-C C1165S (Fig. 1B).…”

Functional and Structural Characterization of the Antiphagocytic Properties of a Novel Transglutaminase from Streptococcus suis

“…Structural analysis revealed that Arg 311 and the catalytic amino acid Cys 302 from one monomer sit on the same ␣-helix (␣7Ј helix), which can be stabilized by water-mediated interactions mediated by ␣4 helix of the other monomer. To our knowledge, similar interactions have not been observed in other solved structures, such as human factor XIII and human TGase 3 (27,29,31). Taken together, we propose that the SsTGase-N monomer was not stable enough to catalyze the reaction in solution environment, whereas dimerization of the protein could promote its activation by stabilizing the architecture of the catalytic cavity.…”

“…Previous studies have mainly focused on the biochemical characteristics of TGases in mammals, but the roles of TGases played in microorganisms remain largely unknown. Among microorganisms, only TGases from Streptoverticillium mobaraense (microbial TGase) and Phytophthora have been studied, and they represent a completely different structure fold compared with those in mammals (27,29). Therefore, bacterial TGases of the PF01841 superfamily are currently largely unknown for their structural features and specific activities.…”

Molecular Basis of Methanol-Inducible Gene Expression and Its Application in the Methylotrophic YeastCandida boidinii

“…Recent analysis of the 3D-crystal structure of TGase has suggested that the pro-peptide hindered access of the substrate to the catalytic site located at the bottom of the cleft. 53) These facts may reflect a regulatory function of the TGase pro-peptide in the original host. Because TGase can be toxic within a bacterial cell, activation of the TGase precursor through processing may be advantageous to avoid negative effects of the synthesized protein within cells, i.e., before secretion.…”