Movements of the Substrate-Binding Clamp of Cypemycin Decarboxylase CypD

Linaridins are a small but growing class of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The class A linaridins, exemplified by cypemycin, possess an unusual S-[( Z)-2-aminovinyl]-d-cysteine (AviCys) residue. Formation of the AviCys in cypemycin requires an oxidative decarboxylation of the precursor peptide C-terminal Cys, and this reaction is catalyzed by a flavin-dependent decarboxylase CypD. In this work, we investigate the molecular recognition processes of CypD by a combination of computational and biochemical analysis. We show that the substrate binding clamp of CypD undergoes dramatic fluctuation, mediating both the substrate entrance into and product release from the catalytic pocket. Extensive molecular dynamic simulations and Fourier transform IR analyses indicated that binding of the substrate induces substantial structural change of the enzyme, converting the substrate-binding clamp from a random loop to a more ordered structure comprising two β sheets and a β turn. The salt bridge between Arg159 guanine and the Cys carboxylate of substrate plays an important role in mediating substrate binding, while hydrophobic interactions are also important in this process. These results provide important mechanistic insights into CypD and other flavin-dependent Cys decarboxylases, and could facilitate future biosynthetic and bioengineering efforts in studying AviCys-containing RiPPs.