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

Abstract

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca2+-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.

Protein Lipidation Catalyzed by Microbial Transglutaminase

“…In designing new lipid substrates for MTG, an MTG-reactive Gln-containing peptidyl substrate was conjugated with fatty acids. Previous studies of peptide screening [11] and docking simulations [12] have indicated that N-terminal hydrophobic residues from the reactive Gln residue was critical for substrate recognition by MTG, and we found that MTG recognizes a seven amino acid sequence (GGGSLLQG) at the C terminus of recombinant proteins. [13] With this in mind, we synthesized lipid-modified peptides (C14-1, C16-1, C18-1, Scheme 1 a) in which the N terminus of the MTG-reactive Gln donor peptide was attached to diverse long-chain fatty acids (myristic acid, palmitic acid and stearic acid) using solid-phase peptide synthesis.…”

Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements

“…Upon examining the crystal structure, we speculated that many of these mutations would react poorly, particularly those located on the internal β‐strands 1 and 4 because they belong to a flat protein surface that does not appear to be complementary to the crevice that forms MTG's active site (Fig. ) . This speculation was invalidated when the most selective and efficient variant was determined to be I6Q, located within β‐strand 1.…”

“…There is a shortage of data indicating which residues play a role in binding MTG's glutamine‐containing substrate. Among the most informative works is an alanine scan of 29 active‐site residues, constituting 9% of the apoenzyme's amino acid sequence . A number of residues were found to be critical for activity, crippling MTG when substituted for alanine.…”

“…Further efforts made to elucidate MTG's mode of substrate recognition include the elucidation of two crystal structures as well as an alanine scan of its broad active site cavity . These have provided a greater understanding of the catalytic mechanism, kinetic parameters, and identifying key residues essential for activity yet did not clarify the characteristics of glutamine reactivity.…”

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

“…Assessing where to direct the P- 8 to P- 10 ligand residues on to the transglutaminase surface may however be challenging. Computational studies have been performed on the docking of inhibitors to FXIII, TGase2, TGase3, and microbial transglutaminase [47; 48; 49]. Similar to ZED1301, the synthetic inhibitors have not extended all the way to the P- 8 to P- 10 substrate positions.…”