Fig. 5

Characterization of LaAIDLs in L. anatina. a Schematic view of LaAIDL1 and 2 with deaminase domains shown in light blue and the catalytic residues in red. b Normalized expression levels of LaAIDL1, 2, and LaRPL39 from the pedicle (Ped), the digestive cecum (DC), the gonad, and the gut from two individuals (La1 and La3) were measured by RT-qPCR with two technical replicates per sample. The error bars indicate the standard deviation. The insets represent magnified views. c–f Deaminase activities of LaAIDL1, LaAIDL2, and mutants of the former were measured in bacterial reversion assays using either a Kanamycin resistance reporter plasmid (c, d) or the endogenous rpoB gene as the reporter (e, f). LaAIDL1 RQ RQAA is putative catalytic mutant in which two residues in both active sites (H81R, E83Q, H298R, and E300Q) were altered. The data for HsAID and empty pASK plasmids is the same as shown in Fig. 3. The horizontal bars represent the median (value below each column), and p-values were calculated for each deaminase compared to empty pASK (c, e), and compared to wildtype LaAIDL1 for (d, f) using a Willcoxon rank sum test for unpaired data. Datasets with p < 0.05 (c: HsAID p = 0.000001 and LaAIDL1 p = 0.000053; d: LaAIDL1 RQRQ p = 0.021; e: HsAID p = 6.6 × 10⁻⁹ and LaAIDL1 p = 0.0022) are marked with asterisks. The insets represent a magnified views of the same data omitting all data points of HsAID (c, e) and one data point of LaAIDL1 RQRQ (f) for clarity. g, h Bacterial expression of V5-tagged LaAIDL1 and 2 (and mutants thereof), and HsAID in E. coli was assessed using a monoclonal anti-V5 antibody (for details see legend to Fig. 3b). Note that the enzymatic activities were tested with untagged versions of the proteins. The predicted sizes for V5-tagged HsAID, LaAID1, and 2 are 25.4, 52.4, and 23.5 kD, respectively, and smaller bands likely represent degradation products