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

Abstract

The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor L-para-aminophenylalanine (L-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster which is reduced to the diferrous state (WT R ) to initiate O 2 activation. However, rapid O 2 activation only occurs 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 O 2 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 O 2 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. Graphical abstract * Corresponding Authors

Unprecedented (μ-1,1-Peroxo)diferric Structure for the Ambiphilic Orange Peroxo Intermediate of the Nonheme N-Oxygenase CmlI

“…The distance associated with the former and its small Debye-Waller factor (σ 2 ) are consistent with the carbon atom from a bidentately bound carboxylate ligand. 58–59, 70 The fact that N = 1 for this scatterer indicates that there must be an average of one bidentate carboxylate per Fe in CmlI R . Also observed is an Fe scatterer at 3.35 Å with a σ 2 of 9.90 × 10 −3 Å 2 .…”

“…The K-edge energy for CmlI R is 7122.1 eV, which falls in the range typical for diferrous species (7121 eV – 7123 eV). 55–58 CmlI Ox has a K-edge energy of 7124.1 eV, which is two electron volts higher than that of the reduced species. This difference in K-edge energy is consistent with the difference observed between diferrous and diferric states in other diiron enzyme systems (Table 1).…”

“…The Fourier transformed (FT) EXAFS data of the various CmlI samples all show an intense feature at R + Δ < 2 Å that is assigned to the scattering atoms of the primary coordination sphere, while the weaker features observed at R + Δ > 2 Å are generally associated with outer sphere interactions such as the other Fe atom in the dinuclear active site as well as the C/N atoms from His residues and carboxylates, as observed in the EXAFS analysis of other nonheme diiron enzymes. 18, 57–58, 68–69 …”

“…Notably, the Fe•••Fe distance at 3.32 Å for CmlI Ox remains essentially the same as that found for the reduced enzyme, a phenomenon that has also been observed for CmlA and hDOHH. 57–58 …”

The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of β-Hydroxylation during Glycopeptide Antibiotic Biosynthesis

“… 23 , 24 Significant insight into the structure and mechanism of both nonheme diiron monooxygenases present in the chloramphenicol biosynthesis pathway—CmlA that is responsible for the β-hydroxylation of PCP-bound 4-aminophenylalanine and CmlI that is responsible for the six-electron oxidation aryl N-oxidation—has been performed. 5 , 18 − 22 , 25 − 32 Given the level of identity shown between CmlA and the related enzymes from GPA biosynthesis (Tcp25/Dbv28/StaM: 34%), 23 , 24 , 33 , 34 along with implicated roles of homologues in both bleomycin (orf3 (His): 39%) 35 , 36 and lysobactin (orf78 (Phe, Leu and Asn): 34%) biosynthesis, 37 this indicated a possibility that CmlA could serve to replace Tcp25 in teicoplanin peptide biosynthesis. One possible caveat was the low identity of the related PCP domains in chloramphenicol and teicoplanin biosynthesis (20%).…”