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.

Structural Characterization of OxyD, a Cytochrome P450 Involved in β-Hydroxytyrosine Formation in Vancomycin Biosynthesis

“…In all cases, the use of the fusion protein was necessary due to the instability of the isolated PCP domain. These results are consistent with those obtained for the binding of PCP-loaded peptides by OxyB, in terms of both spin state change and dissociation constant (17 Ϯ 5 M) (29). They also indicate that only a fraction of the OxyD molecules are able to bind at any one point in time to the PCPloaded substrates.…”

“…Unlike the previously reported successful expression of the PCP6S and PCP7S subunits from the vancomycin NRPS proteins in E. coli (29), the expression of the PCP domain of BpsD and an N-terminally truncated construct were both unsuccessful in E. coli. Attempts to optimize the N-terminal sequence of the BpsD_ PCP were also unsuccessful in yielding any protein, with attempts made to modify the N-terminal portion of the first ␣-helix to resemble that observed in the PCP-7S domain (29). The use of a vector series containing different N-terminal fusion proteins to enhance solubility was attempted (30).…”

“…A synthetic gene encoding a hybrid construct of the C-terminal region of the non-ribosomal peptide synthase BpsD (amino acids 502-581) from A. mediterranei along with the first 14 residues replaced with MEKAPENETEKVLS (the first ␣-helix from the soluble published PCP_7S construct from Amycolatopsis orientalis (29), conserved residues underlined) was obtained from Geneart with concomitant incorporation of unique 5Ј-NdeI and 3Ј-HindIII restriction sites and an N-terminal start codon. The gene was codon optimized for E. coli expression and supplied in the plasmid pMA.…”

Identification of Sesquiterpene Synthases fromNostoc punctiformePCC 73102 andNostocsp. Strain PCC 7120

“…Cell extracts from E. coli transformants expressing either P450NS or P450NP were then added to in vitro assay reactions containing FPP, purified sesquiterpene synthases NP1 or NS1 (depending on the P450 studied) and commercially available spinach ferredoxin and ferredoxin:NADPH reductase to supply reducing equivalents to the P450 enzyme (36). Although the spinach ferredoxin reductase system has been used successfully for an electron donor for other P450s (54), no oxygenated terpene products were detected in these experiments.…”

The Expanding Role of Lipid II as a Target for Lantibiotics

“…The aglycone of each contains the unnatural amino acids hydroxyphenylglycine and dihydroxyphenylglycine, the biosynthetic details of which have recently been described [14]. Cyclization of the peptide backbone was also recently shown to be the result of three unique enzymatic oxidative couplings [15,16]. The resulting polycyclic scaffold provides vancomycin and teicoplanin with a structural rigidity that contributes to their lipid II-binding ability [17][18][19].…”