Characterization of the two-component, FAD-dependent monooxygenase SgcC that requires carrier protein-tethered substrates for the biosynthesis of the enediyne antitumor antibiotic C-1027.
C-1027 is a potent antitumor antibiotic composed of an apoprotein (CagA) and a reactive enediyne chromophore. The chromophore has four distinct chemical moieties, including an ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the biosynthesis of which from l-alpha-tyrosine requires five proteins: SgcC, SgcC1, SgcC2, SgcC3, and SgcC4; a sixth protein, SgcC5, catalyzes the incorporation of this beta-amino acid moiety into C-1027. Biochemical characterization of SgcC has now revealed that (i) SgcC is a two-component, flavin adenine dinucleotide (FAD)-dependent monooxygenase, (ii) SgcC is only active with SgcC2 (peptidyl carrier protein)-tethered substrates, (iii) SgcC-catalyzed hydroxylation requires O 2 and FADH 2, the latter supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre, and (iv) SgcC efficiently catalyzes regioselective hydroxylation of 3-substituted beta-tyrosyl-S-SgcC2 analogues, including the chloro-, bromo-, iodo-, fluoro-, and methyl-substituted analogues, but does not accept 3-hydroxy-beta-tyrosyl-S-SgcC2 as a substrate. Together with the in vitro data for SgcC4, SgcC1, and SgcC3, the results establish that SgcC catalyzes the hydroxylation of ( S)-3-chloro-beta-tyrosyl-S-SgcC2 as the final step in the biosynthesis of the ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety prior to incorporation into C-1027. SgcC now represents the first biochemically characterized two-component, FAD-dependent monooxygenase that acts on a carrier-protein-tethered aromatic substrate.
Lin
Shuangjun
S
Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705-2222, USA.
Van Lanen
Steven G
SG
Shen
Ben
B
D000617:Aminoglycosides / Q000096:biosynthesis* / Q000302:isolation & purification; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis* / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D053281:Enediynes / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
D000617:Aminoglycosides; D000903:Antibiotics, Antineoplastic; D002352:Carrier Proteins; D053281:Enediynes; D011994:Recombinant Proteins; D009243:NAD; C058024:C 1027; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)