Fig. 5: Proposed modes of action of CXE15 and CXE20.

a (i) The NTH (light pink) of CXE15 adopts an open conformation, allowing access for SLs and solvent molecules to enter the catalytic cavity. The loop connecting α7–α8 sits directly above the cavity. Two phenylalanine(s), F89 and F229, extend on either side of the pocket above the catalytic cavity. (ii) In the presence of the hormone, SL (GR24, rectangle, orange), inside the cavity, the NTH closes the pocket. This conformational change results in the reorientation of F89 from an upward to a downward position towards the cavity. Additionally, the loop connecting α7–α8 transitions from a downward to an upward movement, enlarging the pocket’s overall size, and providing more space for GR24 binding. Consequently, M15 from NTH becomes trapped by F89 and F229 through SH–C interactions on each side of the cavity until SL is hydrolyzed. (iii) The SL analog, GR24, undergoes enzymatic action by the catalytic residues S169, H302, and E271, causing the ABC ring’s dissociation from the D-ring. The D-ring (five-membered ring, orange) forms a covalent linkage with S169 and is later modified by the action of CXE15. The fate of the D-ring, whether it undergoes further enzymatic action by CXE15 and the resulting final product, remains unknown and requires further investigation. b (i) The NT α/β segments of CXE20 are adjacent to the catalytic pocket, and the amino acid Y13 extends toward the catalytic cavity. The catalytic cavity of CXE20 is encircled by a ring of phenylalanine(s), and the loop connecting α8–α9 points inward towards the cavity. Unlike CXE15, the pocket of CXE20 is wide open and spacious. (ii) In the presence of the SL, the loop connecting α8–α9 shifts from a downward to an upward position, leading to an increase in the overall size and shape of the pocket. The ring of phenylalanine(s) stabilizes the SL analog GR24 along with Y13 from the N-terminus. (iii) The binding of SL into the cavity results in only very weak hydrolysis, catalyzed by the amino acids S166, H302, and D272 due to the overall increase in the catalytic pocket size. This Figure was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.