Structural characterization of OxyD, a cytochrome P450 involved in beta-hydroxytyrosine formation in vancomycin biosynthesis

Abstract

The cytochrome P450 OxyD from the balhimycin glycopeptide antibiotic biosynthetic operon of Amycolatopsis mediterranei is involved in the biosynthesis of the modified amino acid beta-R-hydroxytyrosine, an essential precursor for biosynthesis of the vancomycin-type aglycone. OxyD binds the substrate tyrosine not free in solution, but rather covalently linked to the carrier protein (CP) domain of the non-ribosomal peptide synthase BpsD, exhibiting micromolar binding affinity to a tyrosine-loaded carrier protein construct. The crystal structure of OxyD was determined to 2.1-A resolution, revealing a potential binding site for the carrier protein-bound substrate in a different orientation to that seen with the acyl carrier protein-bound P450(BioI) (Cryle, M. J., and Schlichting, I. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 15696-15701). A series of residues were identified across known aminoacyl-CP-oxidizing P450s that are highly conserved and cluster in the active site or potential CP binding site of OxyD. These residues appear to be characteristic for aminoacyl-CP-oxidizing P450s, allowing sequence based identification of P450 function for this subgroup of P450s that play vital roles in the biosyntheses of many important natural products in addition to the vancomycin-type antibiotics. The ability to analyze such P450 function based upon sequence data alone should prove an important tool in the analysis and identification of new medicinally relevant biomolecules.

FIGURE 1.

Structures of medicinally important natural products derived from β-hydroxyamino acid residues. Groups derived from the β-hydroxyamino acids are shown in red; novobiocin and clorobiocin also possess an additional moiety derived from a β-hydroxytyrosine, shown in blue.

SCHEME 1.

Formation of l-3-(R)-hydroxytyrosine in A. mediterranei that is required for balhimycin biosynthesis. A, adenylation domain; PCP, peptidyl carrier protein domain; and TE, thioesterase domain.

FIGURE 2.

Titration curves obtained probing the binding of l-tyrosine-loaded Trx-BpsD_PCP (A) and l-tyrosine SNAc to OxyD (B). X-axis, substrate concentration in μm; y-axis, change in the difference in absorbance between 419 nm and 392 nm.

FIGURE 3.

Diagram showing the overall structure of OxyD (glycerol molecules shown in yellow, majority of OxyD shown in gray with certain structural motifs colored for clarity, and heme shown in red) with structural motifs labeled.

FIGURE 4.

A, active site of OxyD showing critical P450 catalytic residues and those interacting with the heme propionate groups, with hydrogen bonding distances indicated (critical catalytic residues shown in magenta, heme interacting residues shown in blue, heme-bound water shown in green, glycerol molecules shown in yellow, heme shown in orange, OxyD shown in gray). B, hydrogen bonding interactions between residues from different structural elements controlling the geometry of the active site pocket, with hydrogen bonding distances indicated (F or G helix residues shown in yellow, I-helix residues shown in cyan, B–B₂ loop residues shown in magenta, β1-sheet residues shown in green, C-terminal loop residues shown in orange, heme shown in red, and OxyD shown in gray).

FIGURE 5.

Conserved residues found in the alignment of PCP-bound amino acid P450s localized on the structure of OxyD. B–B₂ loop residues are shown in magenta, F-helix residues are orange, G-helix residues are yellow, I-helix residues are cyan, β-1 sheet residues are green, heme is dark gray, and OxyD is gray with secondary structure elements labeled.

FIGURE 6.

Docked homology models of the PCP domain of BpsD onto OxyD. OxyD is shown in gray, critical active site residues are colored as in Fig. 6, PCP-model one is cyan, PCP-model two is cyan, and selected OxyD secondary structure elements are labeled; A and B differ in a 45° rotation.

FIGURE 7.

Surface charge of OxyD (A, top view) and docked PCP-models (90° rotation of A into the page; PCP-model one (B), and PCP-model two (C)). OxyD possesses a predominantly negatively charged active site, whereas the PCP domains are both oriented to present a positively charged surface toward OxyD (model of OxyD shown without surface charge for clarity, charged displayed as a surface of blue (positive) and red (negative) charges, OxyD is gray, heme is displayed in gray, PCP-model one is cyan, and PCP-model two is magenta).