title: Identification of the dioxygenase-generated intermediate formed during biosynthesis of the dihydropyrrole moiety common to anthramycin and sibiromycin.
abstract: A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis is a prerequisite for engineering production of analogs with enhanced antitumor activity. Predicted dioxygenases Orf12 and SibV associated with dihydropyrrole biosynthesis in PBDs anthramycin and sibiromycin, respectively, were expressed and purified for activity studies. UV-visible spectroscopy revealed that these enzymes catalyze the regiospecific 2,3-extradiol dioxygenation of l-3,4-dihydroxyphenylalanine (l-DOPA) to form l-2,3-secodopa (λmax=368 nm). (1)H NMR spectroscopy indicates that l-2,3-secodopa cyclizes into the α-keto acid tautomer of l-4-(2-oxo-3-butenoic-acid)-4,5-dihydropyrrole-2-carboxylic acid (λmax=414 nm). Thus, the dioxygenases are key for establishing the scaffold of the dihydropyrrole moiety. Kinetic studies suggest the dioxygenase product is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. The enzymatic product and dimeric state of these dioxygenases are conserved in dioxygenases involved in dihydropyrrole and pyrrolidine biosynthesis within both PBD and non-PBD pathways.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA. Electronic address: ssaha1@umd.edu.
authors.forename: Shalini
authors.initials: S
authors.lastname: Saha
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Gerratana
authors.affiliation: Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA. Electronic address: rokita@jhu.edu.
authors.forename: Steven E
authors.initials: SE
authors.lastname: Rokita
mesh_terms: D000617:Aminoglycosides / Q000737:chemistry* / Q000378:metabolism; D000876:Anthramycin / Q000737:chemistry* / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000617:Aminoglycosides; D011758:Pyrroles; D000876:Anthramycin; C100298:sibiromycin; C013231:pyrroline; D049308:Dioxygenases
keywords: Anthramycin; Biosynthesis; Dihydropyrrole; Extradiol dioxygenase; Pyrrolo[1,4]benzodiazepine; Sibiromycin; l-DOPA; secodopa