title: Oxidative phenol coupling reactions catalyzed by OxyB: a cytochrome P450 from the vancomycin producing organism. implications for vancomycin biosynthesis.
abstract: OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear peptide intermediate linked as a C-terminal thioester to a peptide carrier protein (PCP) domain within the multidomain glycopeptide nonribosomal peptide synthetase (NRPS). Using model peptides with the sequence (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP and (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-(S)Dpg-S-PCP (where Hpg = 4-hydroxyphenylglycine, and Dpg = 3,5-dihydroxyphenylglycine), or containing (R)Leu instead of (R)(NMe)Leu, attached to recombinant PCPs derived from modules-6 and -7 in the vancomycin NRPS, we show that cross-linking of Hpg4 and Tyr6 by OxyB can occur in both hexapeptide- and heptapeptide-PCP conjugates. Thus, whereas OxyB may act preferentially on a hexapeptide still linked to the PCP-6 in NRPS subunit-2, it is possible that a linear heptapeptide intermediate linked to PCP-7 in NRPS subunit-3 may also be transformed into monocyclic product. For turnover, OxyB requires electrons, which in vitro can be supplied by spinach ferredoxin and E. coli flavodoxin reductase. Turnover is also dependent upon the presence of molecular oxygen. The model substrate (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP is transformed into cross-linked product by OxyB with a kcat of 0.1 s-1 and Km in the range 4-13 muM. Equilibrium binding of this substrate to OxyB, monitored by UV-vis, is accompanied by a typical low-to-high spin state change in the heme, characterized with a Kd of 17 +/- 5 muM.
authors.affiliation: Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Katharina
authors.initials: K
authors.lastname: Woithe
authors.forename: Nina
authors.initials: N
authors.lastname: Geib
authors.forename: Katja
authors.initials: K
authors.lastname: Zerbe
authors.forename: Dong Bo
authors.initials: DB
authors.lastname: Li
authors.forename: Markus
authors.initials: M
authors.lastname: Heck
authors.forename: Severine
authors.initials: S
authors.lastname: Fournier-Rousset
authors.forename: Odile
authors.initials: O
authors.lastname: Meyer
authors.forename: Francesca
authors.initials: F
authors.lastname: Vitali
authors.forename: Nobuatsu
authors.initials: N
authors.lastname: Matoba
authors.forename: Khaled
authors.initials: K
authors.lastname: Abou-Hadeed
authors.forename: John A
authors.initials: JA
authors.lastname: Robinson
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D046908:Peptide Biosynthesis, Nucleic Acid-Independent / Q000502:physiology*; D010453:Peptide Synthases / Q000378:metabolism; D010636:Phenols / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014640:Vancomycin / Q000096:biosynthesis*
chemical_list: D010636:Phenols; D014640:Vancomycin; D003577:Cytochrome P-450 Enzyme System; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase