An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway

Thomas M. Makris, Van V. Vu, Katlyn K. Meier, Anna J. Komor, Brent S. Rivard, Eckard Münck, Lawrence Que, John D. Lipscomb

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O2 to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t1/2 = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an 18O2-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η11) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a β(O-O) that is at least 60 cm-1 lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ- η11-peroxo ligand; we propose that a μ- η12-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.

Original languageEnglish (US)
Pages (from-to)1608-1617
Number of pages10
JournalJournal of the American Chemical Society
Volume137
Issue number4
DOIs
StatePublished - Feb 4 2015

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Peptide Synthases
Oxygenases
Biosynthetic Pathways
Chloramphenicol
Peptides
Iron
Enzymes
Ligands
Oxygenation
Streptomyces
Antibiotics
Vibration
Isomers
Vibrations (mechanical)
Paramagnetic resonance
Raman scattering
Absorption spectra
Observation
Electrons
Anti-Bacterial Agents

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An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway. / Makris, Thomas M.; Vu, Van V.; Meier, Katlyn K.; Komor, Anna J.; Rivard, Brent S.; Münck, Eckard; Que, Lawrence; Lipscomb, John D.

In: Journal of the American Chemical Society, Vol. 137, No. 4, 04.02.2015, p. 1608-1617.

Research output: Contribution to journalArticle

Makris, Thomas M. ; Vu, Van V. ; Meier, Katlyn K. ; Komor, Anna J. ; Rivard, Brent S. ; Münck, Eckard ; Que, Lawrence ; Lipscomb, John D. / An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway. In: Journal of the American Chemical Society. 2015 ; Vol. 137, No. 4. pp. 1608-1617.
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abstract = "Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and M{\"o}ssbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O2 to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t1/2 = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an 18O2-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η1:η1) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a β(O-O) that is at least 60 cm-1 lower in energy. M{\"o}ssbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ- η1:η1-peroxo ligand; we propose that a μ- η1:η2-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.",
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T1 - An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway

AU - Makris, Thomas M.

AU - Vu, Van V.

AU - Meier, Katlyn K.

AU - Komor, Anna J.

AU - Rivard, Brent S.

AU - Münck, Eckard

AU - Que, Lawrence

AU - Lipscomb, John D.

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N2 - Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O2 to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t1/2 = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an 18O2-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η1:η1) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a β(O-O) that is at least 60 cm-1 lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ- η1:η1-peroxo ligand; we propose that a μ- η1:η2-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.

AB - Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O2 to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t1/2 = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an 18O2-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η1:η1) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a β(O-O) that is at least 60 cm-1 lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ- η1:η1-peroxo ligand; we propose that a μ- η1:η2-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.

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