Crystal structure of CmlI, the arylamine oxygenase from the chloramphenicol biosynthetic pathway

Abstract

The diiron cluster-containing oxygenase CmlI catalyzes the conversion of the aromatic amine precursor of chloramphenicol to the nitroaromatic moiety of the active antibiotic. The X-ray crystal structures of the fully active, N-terminally truncated CmlIΔ33 in the chemically reduced Fe2+/Fe2+ state and a cis µ-1,2(η1:η1)-peroxo complex are presented. These structures allow comparison with the homologous arylamine oxygenase AurF as well as other types of diiron cluster-containing oxygenases. The structural model of CmlIΔ33 crystallized at pH 6.8 lacks the oxo-bridge apparent from the enzyme optical spectrum in solution at higher pH. In its place, residue E236 forms a µ-1,3(η1:η2) bridge between the irons in both models. This orientation of E236 stabilizes a helical region near the cluster which closes the active site to substrate binding in contrast to the open site found for AurF. A very similar closed structure was observed for the inactive dimanganese form of AurF. The observation of this same structure in different arylamine oxygenases may indicate that there are two structural states that are involved in regulation of the catalytic cycle. Both the structural studies and single crystal optical spectra indicate that the observed cis µ-1,2(η1:η1)-peroxo complex differs from the µ-(η1:η1)-peroxo proposed from spectroscopic studies of a reactive intermediate formed in solution by addition of O2 to diferrous CmlI. It is proposed that the structural changes required to open the active site also drive conversion of the µ-1,2-peroxo species to the reactive form.

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

“…68, 81 The active site picture thus obtained for CmlI R in frozen solution from EXAFS analysis differs significantly from that derived from the crystal structure of CmlI 33 R . 39 For the latter, the hydroxo bridge observed in the EXAFS analysis is absent. Instead, there is a bidentate carboxylate bridge (E326) that gives rise to a longer Fe•••Fe distance of 3.6 Å.…”

“…57–58 These assignments are based on metal-ligand distances found in the crystal structures of nonheme diiron proteins, 7, 70–77 including diferrous CmlI. 39 The shorter Fe–N/O distance at 1.94 Å is assigned to a μ -hydroxo bridge based on comparison to the Fe–O distances associated with the μ -hydroxo ligand in the reduced clusters of CmlA 58 and synthetic ( μ -hydroxo)diferrous complexes. 78–80 …”

“…Recently, the X-ray crystal structures of enzymatic P intermediates have been determined for hDOHH, 38 a truncated CmlI ( CmlIΔ33 ), 39 and T4MO. 35, 40 The hDOHH crystal structure confirms the cis - μ -1,2-peroxo binding mode earlier assigned by resonance Raman studies, although the Fe•••Fe distance in the crystallized hDOHH suggests that this enzyme may have suffered from photoreduction by the X-ray beam, which is a well known issue in metalloenzyme crystallography.…”

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

“…98,289,290 Optical, EPR, Mössbauer, and structural studies have demonstrated conclusively that CmlI, like AurF, contains a di-iron metallocofactor. Also similar to AurF, a combination of UV/Vis, Mössbauer, and resonance Raman spectroscopies have showed that the reaction of reduced Fe 2 II/II ClmI with O 2 generates a peroxo-Fe 2 III/III species ( 110 ) that has spectral properties distinct from those previously characterized in other non-heme di-iron enzymes.…”

Structure/function correlations over binuclear non-heme iron active sites

“…The Mn(III)/ Mn(IV) center can be probed by MCD and the Fe(III) center by NRVS to correlate with the binuclear non-heme Fe enzymes [93]. Finally, a number of new biferrous enzyme classes are emerging (deoxyhypusine hydroxylase (DOHH) [94, 131], urease [95], CmlI [132], etc.) that are accessible to the VTVH MCD/DFT approach presented above to extend Table 1 and develop additional structure/function correlations.…”