SyrB2 in syringomycin E biosynthesis is a nonheme FeII alpha-ketoglutarate- and O2-dependent halogenase.
The nine-residue lipodepsipeptide syringomycin E, elaborated as a phytotoxin by Pseudomonas syringae pv. syringae B301D contains a 4-Cl-L-Thr-9 moiety where failure to chlorinate results in a 3-fold drop in biological activity. The proteins SyrB1 and SyrB2 encoded by the biosynthetic cluster are shown to act as a substrate and enzyme pair for SyrB2-mediated chlorination of the aminoacyl-S-enzyme L-Thr-S-SyrB1. SyrB2 is a member of the nonheme Fe(II) alpha-ketoglutarate-dependent enzyme superfamily, and requires O2 and alpha-ketoglutarate as well as chloride ion to carry out monochlorination of the -CH3 group of L-Thr-S-SyrB1. Chlorination of L-Thr-S-SyrB1 was validated by thioesterase-mediated release of L-Thr and 4-Cl-L-Thr, N-derivatization as fluorescent isoindoles, and HPLC separation compared with authentic standards. Incubations with L-[14C]Thr and [36Cl-] as well as MS of the released products further validated identification. Enzymatic oxidative halogenation is a previously uncharacterized reaction type for nonheme Fe(II) enzymes and may be the general mode for biosynthetic halogenation of aliphatic carbons of natural products.
Vaillancourt
Frédéric H
FH
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Yin
Jun
J
Walsh
Christopher T
CT
D002712:Chlorides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D005296:Ferrous Compounds / Q000378:metabolism; D005453:Fluorescence; D007656:Ketoglutaric Acids / Q000378:metabolism; D013058:Mass Spectrometry; D008956:Models, Chemical; D010100:Oxygen / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D044224:Pseudomonas syringae / Q000201:enzymology*
D002712:Chlorides; D017931:DNA Primers; D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D010456:Peptides, Cyclic; C105246:syringomycin E; D010453:Peptide Synthases; D010100:Oxygen