Extended Data Fig. 8: Proposed mechanism for EasC reaction.
From: Chanoclavine synthase operates by an NADPH-independent superoxide mechanism
a, Mutational results towards the amino acids possibly involving in transferring electron between the haem pocket and the NADPH-binding pocket. The enzyme activity of each sample is calculated as a percentage of the wild type enzyme (WT). The individual (n = 3) and average values along with error bars (s.d.) of each group in one representative experiment among three independent experiments are shown. b, The proposed long-range electron transfer pathway for EasCCf. The Met-Phe-Phe was proposed to be the group of relay amino acids in the process of binding and activation of O2. The distance from those three amino acids to PCC and haem is measured in Å (angstroms). c, Superoxide mediated cooperative catalysis of two pockets in EasC reaction. Upon the binding of O2, PCC in the NADPH-binding pocket donates an electron to Fe(III) in the haem pocket via the relay amino acids M130, F131, and F132 to generate Cpd III and the intermediate I with a radical cation. I was further catalyzed by the superoxide, from the dissociation of CpdIII, to generate II, featuring a hydroperoxide radical. II could equilibrate to the intermediate III with a radical at C10. The hydroperoxide radical in III could mediate the hydrogen abstraction of the carboxylic acid group to initiate the decarboxylation and the following ring cyclization by radical coupling between C5 and C10 to generate IV with a terminal hydroperoxide. The following elimination of H2O2 leads to the formation of CC, aided by hydrogen bond interactions between the hydroperoxide group and surrounding amino acid residues.
