Predicted domain organization and transglutaminase activity of TgpA protein.

(A) Map of the predicted domains DUF3488 (PF11992) and TG (PF01841) along the primary sequence of the PA2873 gene product, called TgpA. The sequence of TgpA spanning aa 396 to 467 of the TG domain is highlighted and aligned to homologous functional TG domains of human coagulation Factor XIII, fish-derived transglutaminase (FTG) and WbmE protein from B. bronchiseptica. Conserved aminoacids of catalytic triad are indicated by an asterisk. (B) Colorimetric assay of transglutaminase activity of purified TgpA TG_180–544 domain by Transglutaminase Colorimetric Microassay Kit (TCM kit; Covalab). TCM kit uses immobilized N-carbobenzoxy(CBZ)-Gln-Gly as the amine acceptor and biotin-conjugated cadaverine as the amine donor. The indicated amounts of purified TgpA TG_180–544 (stock: 2.7 mg/ml, 95% purity) were incubated in 96-well microtiter plate coated with CBZ-Gln-Gly at 37°C for 15 min with calcium, DTT and biotinylated cadaverine, both in the presence and the absence of EDTA supplied in the kit. As a reference for TGase activity, the indicated amounts of kit-included purified guinea pig TGase with specific activity of 0.1 U/mg were incubated under the same conditions. The wells were washed extensively and filled with streptavidin-labelled horseradish peroxidase (HRP) to assay the formation of immobilized γ-glutamyl-cadaverine-biotin by OD₄₅₀ measurement of HRP activity using H₂O₂ as substrate and tetramethyl benzidine as electron acceptor (chromogen).