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-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.

Figure 1

Active site diiron cluster of the diferric CmlA (WT^Ox) from PDB ID 4JO0. Carbon atoms are shown in gray, oxygen atoms in red, nitrogen in blue, and iron atoms are shown as brown spheres.

Figure 2

The diiron cluster observed in the X-ray crystal structure of CmlA in its chemically reduced state (WT^R). (A) Bond distances for the iron and first-sphere ligands, given in Å. (B) Electron density map of WT^R. The blue mesh is the 2|F_o|-|F_c| map contoured at 1.5 σ and the green mesh is the |F_o|-|F_c| omit map for the solvent-derived ligands contoured at +4.5 σ. Atom coloring is as in Figure 1.

Figure 3

XANES region of WT^R (black solid), WT^RU (green dash-dot), WT^RS (blue dotted) and E377D^R (red dashed). Inset: Zoom-in of pre-edge region.

Figure 4

Left: Fourier transform of the unfiltered EXAFS data for WT^R in black, WT^RU in green, WT^RS in blue, E377D^R in red. Right: Unfiltered EXAFS data for CmlA species.

Figure 5

Comparison of the as-isolated UV/vis spectra for WT CmlA (WT^Ox) (black line) and E377D^Ox (red line). Both enzymes are shown at roughly the same concentration based on the absorbance at 280 nm.

Figure 6

Details of the diiron active site observed in the X-ray crystal structure of E377D CmlA in the as-isolated state (E377D^Ox). (A) Bond distances for the iron and first-sphere ligands, given in Å. The mutated residue D377 is starred for clarity. (B) Electron density map of E377D^Ox. The blue mesh is the 2|F_o|-|F_c| map contoured at 1.0 σ and the green mesh is the |F_o|-|F_c| omit map for the μ-oxo bridge and acetate contoured at +4 σ. (C) Overlay of WT^R and E377D^Ox clusters showing the coordination of residue 377. Atom coloring is as in Figure 1 except the carbon atoms of the variant are shown in purple in panel C.

Figure 7

Stopped-flow transient kinetic time courses of the CmlA:CmlP reaction monitored at 340 nm. (A) Reaction of WT^R with CmlP_AT and O₂. With O₂ (black), with CmlP_AT and O₂ (red), and with CmlP_AT~L-PAPA and O₂ (blue). (B) Reaction of E377D^R CmlA with CmlP_AT and O₂. With O₂ (black), with CmlP_AT and O₂ (red), and with CmlP_AT~L-PAPA and O₂ (blue). Reactions were performed in 50 mM HEPES pH 7.5 at 4.5 °C and contained 75 μM CmlA ± 100 μM CmlP_AT and 0.95 mM O₂.

Figure 8

Structural models of WT^R (left) and WT^RS (right) as determined by EXAFS analysis. Numbers in italics represent the best fit scattering distances in Å.

Figure 9

Diiron core differences between CmlA and sMMOH upon redox change. Active site models adapted from crystallographic data from refs⁵, ^{, ,} . Top row: diferrous Fe centers. Bottom row: diferric Fe centers. Left: Fe•••Fe distance contracts while maintaining ∠Fe-O-Fe in sMMOH. Right: ∠Fe-O-Fe increases while maintaining Fe•••Fe distance in CmlA. Distances from EXAFS data from refs¹⁹, ^, . ∠Fe-O-Fe in italics; calculated by assuming a symmetric diiron core, where d(Fe1-O) = d(Fe2-O). Residues shown in red are proposed to shift during the respective catalytic cycles. Both enzymes have μ-1,1-carboxylato residues in the diferrous state (sMMOH E243 and CmlA D403), but only sMMOH E243 is proposed to shift. Some ligands are omitted for clarity.

Figure 10

Proposed structure of O₂ bound peroxo intermediates for sMMOH (left) and CmlA (right). Structures adapted from PDB codes 1FYZ and 5KIK for sMMOH and CmlA, respectively. Residues shown in red are proposed to shift during the respective catalytic cycles. Atoms in blue are from the peroxo moiety, derived from O₂. Some ligands are omitted for clarity.

Figure 11

Computationally docked model of Ppant-L-PAPA in the active site of WT^Ox. The amine group of L-PAPA is within hydrogen bonding distance to the carboxylate of E377. Binding of NRPS~L-PAPA could cause a sterically-driven conformational change of E377 to a monodentate mode which triggers the reaction with O₂. The docked model is based on work presented in ref.⁵