A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis.
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.
Makris
Thomas M
TM
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Chakrabarti
Mrinmoy
M
Münck
Eckard
E
Lipscomb
John D
JD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways; D002701:Chloramphenicol / Q000096:biosynthesis*; D004578:Electron Spin Resonance Spectroscopy; D006900:Hydroxylation; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009876:Operon / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015204:Spectroscopy, Mossbauer; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; C009722:4-aminophenylalanine; D010649:Phenylalanine; D002701:Chloramphenicol; D007501:Iron; D006899:Mixed Function Oxygenases; D010453:Peptide Synthases