Specificity of transglutaminase-catalyzed peptide synthesis

• Wild-type TG2 can catalyze synthesis of Cbz-protected dipeptides.
• Donor substrate specificity is narrow.
• Aromatic ester derivatives of Gly and d-Ala serve as donor substrates.
• Hydrophobic cavity formed by Trp332 and Phe334 dictates substrate specificity.
• Protein engineering based on our computational models may expand substrate scope.

Biocatalytic methods for peptide synthesis are of high value due to the rapidly increasing approval of peptide-based therapeutics and the need to develop new analogs. Guinea pig liver transglutaminase (gTG2) catalyzes the cross-linking of peptides and proteins via the formation of γ-glutamyl-ϵ-lysyl isopeptide bonds. In this study, we investigate gTG2-catalyzed peptide bond formation between various amino acid-derived donor and acceptor substrates. Using LC–MS analysis, we demonstrate that gTG2 forms Gly-Xaa and d-Ala-Gly dipeptide products, confirming that its natural transamidation activity can be co-opted for peptide synthesis. An aromatic ester of Gly was the most efficient acyl-donor substrate tested; aromatic esters of d-Ala and l-Ala showed 50-fold lower reactivity or no reactivity, respectively. A computational strategy combining computational protein design algorithms and molecular dynamics simulations was developed to model the binding modes of donor substrates in the gTG2 active site. We show that the inability of gTG2 to efficiently catalyze peptide synthesis from donors containing alanine results from the narrow substrate binding tunnel, which prevents bulkier donors from adopting a catalytically productive binding mode. Our observations pave the way to future protein engineering efforts to expand the substrate scope of gTG2 in peptide synthesis, which may lead to useful biocatalysts for the synthesis of desirable bioactive molecules.

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