Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis.
The aminocoumarin antibiotics clorobiocin and coumermycin A(1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. l-Proline is selected and activated as l-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CloN5 and CouN5. Enzymatic oxidation of the prolyl-S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product.
Garneau
Sylvie
S
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Dorrestein
Pieter C
PC
Kelleher
Neil L
NL
Walsh
Christopher T
CT
D000595:Amino Acid Sequence; D049933:Aminocoumarins; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000737:chemistry* / Q000302:isolation & purification; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009675:Novobiocin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011758:Pyrroles / Q000737:chemistry*; D015183:Restriction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D049933:Aminocoumarins; D003374:Coumarins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011758:Pyrroles; D009675:Novobiocin; C006260:clorobiocin; C004628:coumermycin