Fig. 3
Biochemical characterization of the Atlantic cod AID’s enzymatic function. A Functional analysis of purified Atlantic cod AID. Gm-AID was expressed and purified alongside other AID homologs as a GST-fusion protein and tested for cytidine deamination activity using the standard alkaline cleavage assay. Deamination activity (% product generated) is presented below each lane. All experiments were done using 32P-labelled TGCbub7 substrate in duplicate. The upper left panel shows a typical alkaline cleavage gel wherein purified Gm-AID and Hs-AID were incubated with substrate at 18, 25, and 37 °C for 16 h showing barely detectible deamination activity for Gm-AID. The upper right panel shows a typical alkaline cleavage gel wherein 16-h prolonged incubation of purified Gm-AID with substrate revealed a preference for lower temperatures. The bottom panel shows a typical alkaline cleavage gel wherein the activity of Gm-AID was tested on 32P-labelled TGCbub7 substrate at various incubation temperature points alongside Hs-AID and Ip-AID of as controls. B The sequence specificity of Gm-AID was compared to that of other AID orthologs. AID was incubated with various substrates containing WRC (TGC, AGC, and TAC) or non-WRC (GGC, GTC, and GAC) motifs at their corresponding optimal temperature. In these experiments, three independent protein preparations were tested for each AID ortholog in duplicate (n = 6). Incubation time was selected based on catalytic robustness of each AID ortholog. Since the absolute activity level on each substrate varied among AID orthologs, relative deamination efficiency was used to enable comparison between AID orthologs. Relative deamination efficiency was calculated by dividing the activity on each substrate by that of the average activity for all 6 studied substrates. Data are represented as mean ± SEM. C Top panel shows the determination of the optimal temperature of Gm-AID compared to that of other AID orthologs at fine temperature increments (4 to 40 °C). Three to six independent protein preparations of each AID ortholog were tested in duplicates. Results are plotted as deamination activity percentage (left panel) and percentage of maximum deamination activity (right panel), revealing optimal temperature of 8, 14, 25, and 31 °C for Gm-AID, Ip-AID, Dr-AID, and Hs-AID, respectively. Data is represented as mean ± SEM. The bottom panel shows time course enzyme kinetics conducted at three temperature points (optimal, below, and above optimal) and corresponding optimal pH of each AID ortholog, in order to confirm the determination of the optimal temperature. Three independent preparations of Gm-AID (30 min to 73 h), Hs-AID (1 min to 70 h), and Dr-AID (30 s to 48 h) were tested in duplicate (n = 6). Data is represented as mean ± SEM