A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase

The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor l-p-aminophenylalanine (l-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster that is reduced to the diferrous state (WT^R) to initiate O₂ activation. However, rapid O₂ activation occurs only when WT^R is bound to CmlP, the NRPS to which l-PAPA is covalently attached. Here the X-ray crystal structure of WT^R is reported, which is very similar to that of the as-isolated diferric enzyme in which the irons are coordinately saturated. X-ray absorption spectroscopy is used to investigate the WT^R cluster ligand structure as well as the structures of WT^R in complex with a functional CmlP variant (CmlP_AT) with and without l-PAPA attached. It is found that formation of the active WT^R:CmlP_AT-l-PAPA complex converts at least one iron of the cluster from six- to five-coordinate by changing a bidentately bound amino acid carboxylate to monodentate on Fe1. The only bidentate carboxylate in the structure of WT^R is E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O₂ in the presence or absence of CmlP_AT-l-PAPA, showing loss of regulation. However, this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O₂ reactivity with juxtaposition of the substrate and the reactive oxygen species. The carboxylate shift in response to substrate binding represents a novel regulatory strategy for oxygen activation in diiron oxygenases.