Fig. 5: AKRtyl binds two tylosins and tylosin^a functions as allosteric modulator.

a Side views of the AKRtyl octamer binding to two tylosins (tylosin^o: orthomeric tylosin, green sphere; tylosin^a: allosteric tylosin, yellow sphere). b Magnified section showing the tylosin^o and its binding site. The tylosin^o is shown as green balls-and-sticks, the interacting residues are shown as blue or red (another chain) sticks, and the NADP⁺ is shown as white balls-and-sticks in the back. The tylosin^o structural model is superposed with the corresponding 2F_o-F_c electron density map contoured at 0.7σ (black). Tyr55 and His130 form hydrogen bond interactions (light purple dotted lines) with the aldehyde group of the substrate. c Close-up view of the binding mode of the tylosin^a on the allosteric site (α-helices 7 and 10). The tylosin^a is shown as yellow balls-and-sticks. The structural model is superposed with the corresponding 2F_o-F_c electron density map contoured at 1.0σ (black). The closest distance from the tylosin^a to the tylosin^o and NADP⁺ are 10.1 Å and 14.9 Å, respectively. d, e The disaccharide side and macrolide side of the allosteric site and the superposition of the tylosin^a bound (blue) and unbound (gray). The interacting residues are shown as sticks and the hydrogen bonding interactions are shown as light purple dotted lines. Glu193 and Arg195 forming hydrogen bonding interactions with the hydroxyl group at the C4 position of α-L-mycarose and Glu253 and Arg257 with the aldehyde group at the C20 position on tylosin and others are mainly non-polar hydrophobic interactions.