Oxidative Phenol Coupling Reactions Catalyzed by OxyB: A Cytochrome P450 from the Vancomycin Producing Organism. Implications for Vancomycin Biosynthesis
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 with...
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Published in | Journal of the American Chemical Society Vol. 129; no. 21; pp. 6887 - 6895 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
WASHINGTON
American Chemical Society
30.05.2007
Amer Chemical Soc |
Subjects | |
Online Access | Get full text |
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Summary: | 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 k cat of 0.1 s-1 and K m in the range 4−13 μM. 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 K d of 17 ± 5 μM. |
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Bibliography: | istex:3F497D7769120A25FF1C53EBA69554894725FC1A ark:/67375/TPS-J5B8P4ZQ-K ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0002-7863 1520-5126 |
DOI: | 10.1021/ja071038f |