Fig. 5. A combination of residues from the active site, loop, and tunnel region are responsible for the selectivity and substrate scope of the SxtT catalyzed reaction.

a The effect of combining residue variants from the active site (M255Y, T276V), loop (R204K), and tunnel region (W214M/S232A/I237T) was tested using ddSTX, β-STOH, and STX as substrates. b The position of hydroxylation on a β-STOH substrate was confirmed using an ethanol-incorporation experiment. The essential lack of an ethanol-derivatized product in the active site + loop + tunnel chimera variant (99% of hydroxylation at C11) suggested that selectivity had been changed to resemble GxtA (solid colors). The position of hydroxylation when α-STOH was used as a substrate was tested in the tunnel chimera and the active site + loop + tunnel chimera variants (patterned colors). Both variants, like GxtA, showed a preference for hydroxylating at the C11 position. In the tunnel chimera, using an α-STOH substrate, 98% of the product is hydroxylated at the C11 position, whereas in the active site + loop + tunnel chimera variant, 100% of the product showcases a hydroxyl group at the C11 position. c The ability to hydroxylate naturally occurring saxitoxin analogs (top panel) was evaluated for the active site + loop, tunnel chimera, and the active site + loop + tunnel chimera variants. The activity is shown as a heat map with the lowest activity in white and the highest activity in dark teal. The percent conversion of the native substrates of SxtT and GxtA are shown in white. In panel a, data was measured using n = 2 independent experiments. In panels b and c, the data were also measured using n = 2 independent experiments and is presented as the mean value of these measurements. In panels a and c, ddSTX corresponds to dideoxysaxitoxin, β/α-STOH corresponds to β/α-saxitoxinol, and STX corresponds to saxitoxin. The abbreviation dc-represents decarbamoyl. Source data are provided as a Source Data file.