Fig. 3. Identification of a second structural region in SxtT and GxtA that is involved in selectivity.

Different conformations of a flexible loop that connects the β13 and β14 strands of a SxtT and b GxtA are observed in each substrate-bound structure. In SxtT this loop (green) interacts with the substrate as well as Ser230 and Glu272. In GxtA, this flexible loop is found in two different orientations (pink and wheat). The difference between the position of Lys204 on these loops is 9.8 Å. c The pink loop orientation shown in panel b interacts with residues Ser233 and Glu234 on the surface of the protein. d A surface rendering of SxtT with β-STOH bound shows that the loop (green) closes the entry (blue) into the active site. e A similar surface rendering of GxtA reveals that the different observed loop orientations (pink and wheat) extend away from the active site and leave the entrance (blue) wide open. f A chimeric variant in the loop region of SxtT (F200V, Q201H, R204K, I205F, V206N, S207N, H208S, I209T, E210K, and W214M) shows low levels of activity on ddSTX and β-STOH, whereas the single R204K loop variant is able to hydroxylate ddSTX, β-STOH, and STX. Similarly, the creation of a triple variant that combines residue changes at the R204 position with changes in the active site region (M255Y and T276V) amplifies the ability to hydroxylate STX. g As evidenced by incorporation of ethanol into the reaction product, a mixture of hydroxylation at the C11 (purple) and C12 (brown) positions is observed with the R204K and M255Y/T276V/R204K variants when β-STOH is used as a substrate. In panels f and g, data were measured using n = 2 independent experiments. In panel g, data are presented as the mean value of the measurements. In all panels, ddSTX corresponds to dideoxysaxitoxin, β-STOH corresponds to β-saxitoxinol, and STX corresponds to saxitoxin. Source data are provided as a Source Data file.