Publications

• A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis.

  Makris T.M., Chakrabarti M., Muenck E., Lipscomb J.D.

  The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide sy ... >> More

  The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mössbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics. << Less

  Proc. Natl. Acad. Sci. U.S.A. 107:15391-15396(2010) [PubMed] [EuropePMC]

• Diiron monooxygenases in natural product biosynthesis.

  Komor A.J., Jasniewski A.J., Que L., Lipscomb J.D.

  Covering: up to 2017 The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only recently. At present, two families have been discovered. The archetypal member of the first family, CmlA, catalyzes β-hydroxylation ... >> More

  Covering: up to 2017 The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only recently. At present, two families have been discovered. The archetypal member of the first family, CmlA, catalyzes β-hydroxylation of l-p-aminophenylalanine (l-PAPA) covalently linked to the nonribosomal peptide synthetase (NRPS) CmlP, thereby effecting the first step in the biosynthesis of chloramphenicol by Streptomyces venezuelae. CmlA houses the diiron cluster in a metallo-β-lactamase protein fold instead of the 4-helix bundle fold of nearly every other diiron monooxygenase. CmlA couples O2 activation and substrate hydroxylation via a structural change caused by formation of the l-PAPA-loaded CmlP:CmlA complex. The other new diiron family is typified by two enzymes, AurF and CmlI, which catalyze conversion of aryl-amine substrates to aryl-nitro products with incorporation of oxygen from O2. AurF from Streptomyces thioluteus catalyzes the formation of p-nitrobenzoate from p-aminobenzoate as a precursor to the biostatic compound aureothin, whereas CmlI from S. venezuelae catalyzes the ultimate aryl-amine to aryl-nitro step in chloramphenicol biosynthesis. Both enzymes stabilize a novel type of peroxo-intermediate as the reactive species. The rare 6-electron N-oxygenation reactions of CmlI and AurF involve two progressively oxidized pathway intermediates. The enzymes optimize efficiency by utilizing one of the reaction pathway intermediates as an in situ reductant for the diiron cluster, while simultaneously generating the next pathway intermediate. For CmlI, this reduction allows mid-pathway regeneration of the peroxo intermediate required to complete the biosynthesis. CmlI ensures specificity by carrying out the multistep aryl-amine oxygenation without dissociating intermediate products. << Less

  Nat Prod Rep 35:646-659(2018) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of a dinuclear iron cluster-containing beta-hydroxylase active in antibiotic biosynthesis.

  Makris T.M., Knoot C.J., Wilmot C.M., Lipscomb J.D.

  A family of dinuclear iron cluster-containing oxygenases that catalyze β-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Münck, E., and Lipscomb, J. D. (2010) Proc. Natl. Aca ... >> More

  A family of dinuclear iron cluster-containing oxygenases that catalyze β-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Münck, E., and Lipscomb, J. D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 15391-15396]. Here, the 2.17 Å X-ray crystal structure of the archetypal enzyme from the family, CmlA, is reported. CmlA catalyzes β-hydroxylation of l-p-aminophenylalanine during chloramphenicol biosynthesis. The fold of the N-terminal domain of CmlA is unlike any previously reported, but the C-terminal domain has the αββα fold of the metallo-β-lactamase (MBL) superfamily. The diiron cluster bound in the C-terminal domain is coordinated by an acetate, three His residues, two Asp residues, one Glu residue, and a bridging oxo moiety. One of the Asp ligands forms an unusual monodentate bridge. No other oxygen-activating diiron enzyme utilizes this ligation or the MBL protein fold. The N-terminal domain facilitates dimerization, but using computational docking and a sequence-based structural comparison to homologues, we hypothesize that it likely serves additional roles in NRPS recognition and the regulation of O2 activation. << Less

  Biochemistry 52:6662-6671(2013) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.