Fig. 2. Detection of Dpa enzyme substrate.

a Protein (left), RNA (center) and small molecule (right) extracts from exponential phase of A. baumannii cells cultured in liquid LB media were subjected to acetylation assays with purified Dpa enzyme and isotope labelled [14 C]acCoA. Proteins were then migrated on SDS-PAGE and stained with Coomassie; RNA was migrated on PAGE gel and stained with methylene blue. Small molecules were migrated on thin layer chromatography plate. Gels and TLC plates were dried, and isotope signals were revealed with autoradiography. Results were reproduced three times independently; representative gels and plates are shown. b Small molecules from A. baumannii cellular extract and commercial polyamine substrates: 1,3-diaminopropane (1,3-DAP), putrescine (PUTR.), cadaverine (CADAV.), spermidine (SPMD) and spermine (SPMN) were subjected to acetylation assays with purified Dpa enzyme and isotope labelled [14 C]acCoA for 30 min at 37 °C. Reactions were migrated on TLC plate and revealed by autoradiography. Structural formulas of polyamines are indicated on top of the gel. c Acetylation of polyamines by bacterial GNAT acetyltransferases – A. baumannii Dpa, E. coli SpeG, A. baumannii Hpa2 and S. enterica Aac(6’)-Iy. Final concentration of 5 mM of different polyamines were incubated with 0.5 mM of acCoA and 2 µM of enzymes for 30 min at 30 °C and acetylation was revealed with DTNB reagent as described in the methods section. d Acetylation of 1,3-DAP as compared to mono-acetylated 1,3-DAP (N-(3-aminopropyl)acetamide) indicated as N-(3-AP)AA. Reactions were performed as mentioned above, except that an increasing amount of N-(3-AP)AA was pre-mixed with the enzyme before the reaction (when indicated), otherwise 5 mM of substrates were used. Each reaction was performed three times over independent experiments, error bars show standard deviation. Source data are provided as a Source Data file.