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Originally published In Press as doi:10.1074/jbc.M306486200 on July 29, 2003

J. Biol. Chem., Vol. 278, Issue 47, 46727-46733, November 21, 2003
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Crystal Structure of OxyC, a Cytochrome P450 Implicated in an Oxidative C–C Coupling Reaction during Vancomycin Biosynthesis*

Olena Pylypenko{ddagger}§, Francesca Vitali¶, Katja Zerbe¶, John A. Robinson¶||, and Ilme Schlichting{ddagger}§**

From the {ddagger}Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund, Germany, §Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany, and Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

Received for publication, June 18, 2003 , and in revised form, July 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Gene inactivation studies point to the involvement of OxyC in catalyzing the last oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. Presently, the substrate and exact timing of the OxyC reaction are unknown. The substrate might be the bicyclic heptapeptide or a thioester derivative bound to a protein carrier domain. OxyC from the vancomycin producer Amycolatopsis orientalis was produced in Escherichia coli and crystallized, and its structure was determined to 1.9 Å resolution. OxyC gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. The addition of vancomycin aglycone to OxyC produced type I changes to the UV spectrum. OxyC exhibits the typical P450-fold, with the Cys ligand loop containing the signature sequence FGHGX-HXCLG and Cys-356 being the proximal axial thiolate ligand of the heme iron. The observation of a water molecule bound to the heme iron is consistent with the UV-visible spectra of OxyC indicating a low spin heme. A polyethylene glycol molecule occupying the active site might mimic the bicyclic heptapeptide substrate. Analysis of the structure of Oxy-proteins and other P450s indicates regions that might be involved in binding of the redox partner and possibly the protein carrier domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Vancomycin and related glycopeptides are clinically important antibiotics that act as inhibitors of bacterial peptidoglycan biosynthesis (1, 2). These antibiotics typically possess a heptapeptide aglycone that is rigidified by side chain cross-linking involving several of its constituent aromatic amino acids (Fig. 1). As a result of this cross-linking, a molecular cavity is created within the glycopeptide core, the surface of which is complementary to that of the terminal D-Ala-D-Ala dipeptide unit present in intermediates of peptidoglycan biosynthesis (2). The glycopeptides are thus able to bind this dipeptide with high affinity. The aromatic cross-links arise formally through oxidative phenol coupling reactions. Although phenol coupling reactions are widespread in natural product biosynthesis as a whole, knowledge about the enzymes catalyzing such processes is sparse. It is clear, however, that members of the cytochrome P450 family have evolved that are able to promote specific phenol coupling reactions, both in glycopeptide antibiotic biosynthesis and in other areas of secondary metabolism, particularly alkaloid biosynthesis (3).



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  		FIG. 1.
Structures of glycopeptide antibiotics.

 
Biosynthetic gene clusters for several glycopeptide antibiotics have been cloned and sequenced recently, including those for chloroeremomycin (4), balhimycin (5), and teicoplanin (6) production. The organization of the biosynthetic genes in each cluster is very similar. This includes in the chloroeremomycin cluster three large nonribosomal peptide synthetase (NRPS)1 genes and three P450 genes (oxyA, oxyB, and oxyC), as well as other genes involved in the biosynthesis of the constituent amino acids (4). Using probes based on the vancomycin gtfE biosynthetic gene, a region homologous to three P450 genes (oxyA, oxyB, and oxyC) has also been cloned from the vancomycin producer Amycolatopsis orientalis (7). The involvement of the OxyA–C gene products in the three phenol coupling reactions required for balhimycin production, as well as the order of the coupling steps, has been established by gene knock-out experiments (8, 9). The first coupling should occur between residues 4 and 6 catalyzed by OxyB, the second between residues 2 and 4 catalyzed by OxyA, and the last between residues 5 and 7 catalyzed by OxyC. Whereas the first two are C–O couplings, the last catalyzed by OxyC involves a C-C coupling. However, it is not yet known whether the cross-linking steps occur on free heptapeptide intermediates or while the peptide is still attached as a thioester to a peptide carrier domain (PCD) of the NRPS. The latter has been found for instance for the P450 enzyme NovI, which catalyzes the {beta}-hydroxylation of tyrosine during novobiocin biosynthesis; it hydroxylates a tyrosine moiety that is coupled as a thioester to a peptide carrier domain (10). A similar route for the production of a {beta}-hydroxytyrosine moiety may also occur during vancomycin biosynthesis. So far, no reports of in vitro activity for the enzymes OxyA–C have been described. Recent attempts to demonstrate conversion of putative linear heptapeptide intermediates by OxyB failed to give significant amounts of any new cross-linked products resulting from oxidative coupling (7). Presently, the optimal substrate for OxyC is also unknown, but it should be a doubly cross-linked heptapeptide either in a free form or possibly linked as a thioester to a PCD. This thioester, after transformation on OxyC and hydrolytic cleavage of the C-terminal thioester, would afford vancomycin aglycone.

The influence of the chlorine atoms in residues 2 and 6 on the rates of cross-linking also has not been established. However, nonchlorinated cross-linked intermediates have been isolated from gene knock-out mutants of the balhimycin producer (8), showing that the chlorine atoms are not essential for cross-coupling to occur in this pathway.

In this publication, we report the purification, preliminary characterization, and crystal structure of OxyC, generated from the vancomycin oxyC gene, and its comparison with OxyB from the same producer. The crystal structure of OxyC is presented with the reservation that no enzymatic activity has so far been demonstrated with the protein. The potential substrates, a doubly cross-linked free heptapeptide or one bound to a PCD, are so far not available to test enzymatic activity. All attempts here to co-crystallize OxyC with vancomycin aglycone failed to produce crystals of the complex. However, the crystal structure of OxyC does reveal a well ordered polyethylene glycol (PEG) molecule bound in the presumed active site of the enzyme.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Protein Production—A. orientalis DSM40040 chromosomal DNA was the source of oxyC, as described earlier (7). The oxyC gene was amplified by PCR using the following primers (restriction sites are underlined): OxyCFor, 5'-GCTATCTAGACATATGGGTCACGATATCGATCAGGTCG-3'; OxyCRev, 5'-TGATCAAGCTTAGGCCTTCACCAGGTAACCGGTACCTGA-TCAGGGC-3'.

The PCR product was digested with XbaI and HindIII and ligated into XbaI/HindIII-digested pBluescript for nucleotide sequencing. After the correct nucleotide sequence was confirmed, the gene was transferred as an NdeI/HindIII fragment into pET17b (Novagen) to give pOCI811. For the production of His6-tagged OxyC (His6-OxyC), the gene was transferred from pOCI811 as an NdeI/XhoI fragment into pET14b to give pOCI1048.

In this work, OxyC was produced with a native N terminus, and as a fusion protein with an N-terminal His6 tag (called here His6-OxyC). For the production of both proteins, the same procedures were used as for OxyB as reported earlier (7). After purification, both proteins showed a single band by SDS-PAGE, gave an electrospray mass spectrum consistent with the expected mass and the correct N-terminal sequence by automated Edman degradation (data not shown). For crystallization, the His6 tag was removed by thrombin cleavage. The cleavage step was done before the anion-exchange chromatography step. His6-OxyC (1.75 mg/ml) was dialyzed in thrombin cleavage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2.5 mM CaCl2) and incubated overnight at 30 °C with 1 unit of thrombin/10 mg of protein. Then the protein was applied to a nickel-nitrilotriacetic acid column, and flow-through fractions containing OxyC were collected.

UV-visible Spectrophotometry—All spectra were obtained with a Varian Cary 3 double-beam spectrophotometer equipped with thermostated cell holder. CO difference spectra of reduced OxyC were measured as described earlier for OxyB (7). The imidazole complex with OxyC was generated by adding a buffered solution of imidazole to a final concentration of 50 mM to a solution of OxyC (2.5 µM) in 50 mM Tris-HCl at pH 8.0. To monitor the binding of vancomycin aglycone, a solution of enzyme (3.1 µM) in 50 mM Tris-HCl (2.0 ml, pH 8.0) was divided between two tandem cuvettes. After thermal equilibration at 30 °C the base line was zeroed between 300 and 700 nm. Successive aliquots of vancomycin aglycone dissolved in 10% methanol in H2O were added to the sample cuvette to give the final ligand concentration in the range of 500 µM. The methanol concentration did not exceed 1% v/v. An equal volume of 10% methanol in H2O was added each time to the reference cuvette, and the difference spectrum was recorded. The binding of PEG was assayed in a similar way.

Crystallization of OxyC—Crystals were obtained at 20 °C using the hanging drop method by mixing 1 µl of protein (15 mg/ml in a solution containing 20 mM KCl, 5 mM dithioerythritol, 20 mM K-Hepes, pH 7.0) and reservoir solutions (0.1 M di-ammonium tartrate, 5 mM dithioerythritol, 100 mM K-Hepes, pH 7.5, and 12% PEG 550 monomethyl ether) using microseeding. Prior to flash-cooling in liquid nitrogen, the crystals were rinsed briefly in the cryoprotectant solution consisting of the reservoir solution but with 30% PEG. The crystals contain two molecules of OxyC per asymmetric unit and have the symmetry of the monoclinic space group P21 with unit cell parameters a = 41.1 Å, b = 99.3 Å, c = 105.1 Å, {beta} = 96.11°.

X-ray Data Collection and Structure Determination—Crystals were kept at 100 K during measurements. Diffraction data were collected using a rotating anode or synchrotron radiation (beamline ID14–1 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France) and processed with the XDS suite of programs (11) (see Table I). The OxyC structure was determined by molecular replacement using the program AMORE (12). A homology model derived from the crystal structure of OxyB (Protein Data Bank code 1LFK [PDB] ) was used as a search model. Two molecular replacement solutions corresponding to the two molecules in the asymmetric unit were found. The resulting phase information allowed us to build the missing parts of the protein model using the program O (13). The refinement (including simulated annealing) was performed using crystallography NMR software (14, 15) and was monitored by Rfree, computed with 5% of the data randomly selected and omitted from the refinement. During several rounds of refinement and manual rebuilding, solvent molecules were included in the model using the WATERPICK routine of crystallography NMR software (14). All waters were checked manually and removed if displaying unusual H-bonding geometry. The final model contains 780 residues, 603 water molecules, and 4 PEG molecules. Three parts of the OxyC structure (molecule A: 1–4, 81–89, 184–186; molecule B: 1, 77–90, 184–185) are too disordered to be built into the electron density map (see Table I). Coordinates and structure factors have been deposited with the Protein Data Bank (Protein Data Bank code 1UED).


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  		TABLE I
Statistics of diffraction data and refinement

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Production of OxyC—OxyC with a native N terminus (native OxyC) and with an N-terminal His6 tag (His6-OxyC) were produced in Escherichia coli using the methods reported earlier for OxyB (7). His6-OxyC and native OxyC gave essentially identical UV-visible absorption spectra that are typical of P450 hemoproteins (16). All of the UV spectra reported below are with the native OxyC, whereas His6-OxyC with cleaved His6 tag afforded crystals that were used for the structure determination.

UV-visible Spectroscopy—As shown in Fig. 2, the UV-visible absorption spectrum of OxyC has a Soret band at 417 nm, and {beta} and {alpha} peaks at 542 and 567 nm, respectively, typical for those of low spin cytochrome P450s. Reduction with Na2S2O4 shifts the Soret band to 418 nm and the {beta} and {alpha} peaks to 534 and 560 nm. Subsequent addition of CO gives a characteristic CO difference spectrum showing a prominent peak at 450 nm. These results show that the isolated OxyC is in the oxidized form and can be reduced with sodium dithionite.



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  		FIG. 2.
UV-visible absorption spectra of OxyC in Tris-HCl buffer (50 mM, pH 8.0) at 30 °C. A, the spectrum of OxyC is shown in red, in blue after reduction with dithionite, and in green after the addition of imidazole (50 mM). B, the CO-difference spectrum of reduced protein showing the characteristic P450 band.

 
The addition of imidazole to the oxidized form causes a typical red shift in the Soret band to 422 nm and {beta} and {alpha} peaks at 541 and 569 nm (Fig. 2). The OxyC proteins with and without the His tag behaved identically, suggesting that the His6 tag does not perturb the heme environment or provide an imidazole ligand for the iron. Indeed, the crystal structure of OxyC shows clearly a bound water molecule in the sixth coordination position (see below). Fig. 3 shows difference UV spectra obtained with OxyC and increasing concentrations of imidazole. This type II binding spectrum reflects increased occupancy of the axial ligand site by imidazole. The data show that the binding site is saturable at high imidazole concentrations (>20 mM).



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  		FIG. 3.
UV-visible difference spectra of OxyC upon addition of increasing concentrations of imidazole (0, 1, 4, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mM imidazole).

 
UV-visible difference spectra recorded with OxyC and increasing amounts of vancomycin aglycone (Fig. 4) showed a trough at {approx}418 nm, and an increase at <=400 nm, changes similar to those caused by a type I ligand. It is known for many P450s that type I ligands, mostly substrates and exogenous ligands, can cause a shift in the Soret band from 418 nm to 388–394 nm (in the UV difference spectrum there is a peak at {approx}385 nm and a trough at {approx}420 nm) upon binding, indicating the displacement of the axial ligand from the heme-Fe atom with a change in heme spin state from hexacoordinated low spin to pentacoordinated high spin. The concentration dependence of the spectral changes (Fig. 4) shows that the binding site is not saturated even at the highest concentrations of vancomycin aglycone tested here (~0.5 mM), indicating a rather weak interaction. Attempts to co-crystallize OxyC with the vancomycin aglycone failed.



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  		FIG. 4.
UV-visible difference spectra of OxyC upon addition of increasing concentrations of vancomycin aglycone (0, 100, 200, 300, 400, and 500 µM).

 
Crystallization—Screening of random crystallization conditions with His6-OxyC did not result in any positive results, whereas OxyC with cleaved His6 tag gave microcrystals. Further optimization of crystallization conditions in combination with microseeding resulted in crystals with two different morphologies. Crystals with square cross-sections had diffraction patterns indicating a super-lattice; only the form with hexagonal cross-sections gave interpretable diffraction patterns. A 1.9-Å data set was collected at the European Synchrotron Radiation Facility; data statistics are listed in Table I.

Structure Determination—The high homology of the OxyC and OxyB primary structures (45% identity and 58% similarity) allowed structure determination by molecular replacement. A homology model derived from the OxyB structure (7) using the program O (13) was used as a search model. Two solutions were found representing the two molecules of OxyC in the asymmetric unit.

Overall Structure—OxyC exhibits the typical P450-fold and has high structural similarity to OxyB (7) (r.m.s.d. 1.9 Å). Compared with OxyB, the OxyC N terminus is 8 residues longer; these residues are folded as an additional {beta}-strand ({beta}10) in {beta}1 similar to P450cam (17) (Fig. 5). In all known P450 structures the {beta}11 and {beta}12 strands are connected by a flexible loop. It has a three-amino acid residue insertion in OxyC and forms two turns of an {alpha}-helix (A''). The structures of the C termini of OxyC and OxyB differ also. In the former, there is an additional {alpha}-helix M followed by strand {beta}33 and the region corresponding to {beta}4 in OxyB has a loop conformation.



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  		FIG. 5.
Stereo view of a ribbon representation of the OxyC structure. Secondary structure elements different in OxyC and OxyB are shown in yellow. The figure was prepared using MOLSCRIPT (30) and RASTER3D (31, 32).

 
As shown in Fig. 6 the structures of the two molecules in the asymmetric unit are rather similar (r.m.s.d. 0.32 Å) except for the more flexible regions such as the F-helix and the N-terminal and C-terminal ({beta}4 region, {beta}32 and the M-helix) parts (r.m.s.d. including these regions0.45 Å).



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  		FIG. 6.
Stereo view of superimposed C-{alpha} traces of the two crystallographically independent copies of the OxyC (blue, molecule A; magenta, molecule B). The numbers refer to amino acid residues. The transformations for optimal superposition were determined from equivalent C-{alpha} atoms using molecule A as a reference and then applied to the heme and the PEG molecule as well. Figs. 6,7,8 and 10 were prepared using WebLab ViewerLite 4.0 (Molecular Simulations Inc.).

 
The structure starts with Gly-2 and there is no A'-helix. Similar to OxyB (7) OxyC contains a {beta}-hairpin, {beta}0. This structural element, which seems to be a common feature of oxyproteins, is formed by a patch of amino acid residues that are conserved in oxy-proteins but not in all P450s (Fig. 7, green). As in the OxyB structure the main chain atoms of Arg-21 (identical) located in this hairpin are solvent-accessible, its side chain is turned toward the inside of the molecule, and the guanidinium group forms hydrogen bonds with the carbonyl oxygen atoms of Leu-289 (identical) and Ser-290 (homologous). This interaction seems to stabilize Ser-290 in an energetically strained conformation in which its hydroxyl group interacts with the carbonyl of Arg-287 and the side chain amide of Asn-325. The residues following Ser-290, Pro-291, and Val-292 belong to the region connecting the K-helix and {beta}14 and form a "wall" of the substrate-binding pocket. Moreover, Val-292 is in van der Waals contact with Thr-249, which is assumed to be catalytically important and may be required for positioning of the latter residue.



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  		FIG. 7.
Stereo view of the OxyC C-{alpha} trace. The three patches of amino acid residues conserved among oxy-proteins but not among all P450s are shown in different colors.

 
The most variable parts in the P450 primary structures correspond to the B'-, F- and G-helix regions, which are known to be important in substrate binding (18). There is no electron density for most of the B'-helix in OxyC, and the residues between Gln-79 and Ala-91 are not visible; the data indicate a region of high flexibility in the protein similar to OxyB. The lengths and orientations of the F- and G-helices are similar in OxyB and OxyC, they resemble the substrate-free "open" conformation of P450-BM3. In OxyC, the loop connecting the F- and G-helixes (Asp-184 to Ser-186) is too disordered to be modeled. This may be because of high flexibility of the region or to protein degradation. This region contains the sequence RRNADLSRG, which may be recognized and cleaved by the protease thrombin used for His tag cleavage during the protein purification.

The F-helix in OxyC is formed by an arginine-rich sequence. Thus, there is a cluster of positively charged residues on the surface of the F-helix extending to the G-helix (Fig. 8). The second patch of four Arg residues (three of them are conserved among oxy-proteins) is located on the D-helix. This region has been implicated in the binding of the redox partner in the crystal structure of the complex of the heme and FMN domains of P450-BM3 (19). It is known that in flavodoxins and cytochrome P450 reductase (CPRFMN) (20), conserved negatively charged amino acid residues are clustered near the flavin; Cys4Fe2S2 ferredoxins also have acidic surfaces at the [Fe2S2] cluster site (21). Thus, the positively charged cluster in OxyC might be involved in interactions with a redox partner by electrostatic attraction of complementary negatively charged surfaces.



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  		FIG. 8.
Arginine-rich patches on the OxyC surface.

 
The Active Site—The most highly conserved regions in the P450 superfamily are near the heme, and include the I- and L-helices, and the heme Cys-ligand loop. The latter contains the P450 signature sequence FGHGXHXCLG, with Cys-356 being the proximal axial thiolate heme ligand in OxyC. The I-helix spans the whole molecule and is located above the heme (see Fig. 9A). It contains two conserved catalytically important residues, an acidic residue (Glu-248) followed by a threonine (Thr-249). This region is believed to be involved in the proton relay shuttling protons from the solvent to the heme bound oxygen molecule during catalytic turnover, (22, 23). The Metwater molecule (Wat-25) bound to the heme iron forms hydrogen bonds with the carbonyl oxygen of Gly-246. The latter interaction is similar to the corresponding one in OxyB (7), P450terp (24), and substrate free P450-BM3 (25), and stabilize the iron bound water molecule (see Fig. 9A). In P450-BM3, substrate binding induces a conformational change in the I-helix resulting in a shift of Ala-264 and a concomitant movement of the Met-water to a new position thus allowing binding of oxygen to the heme iron (26). A similar situation can be expected for OxyC.



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  		FIG. 9.
A, stereo view of the distal heme pocket of OxyC showing the conformation around Thr-249. The sigmaA-weighted 2Fo – Fc electron density map is contoured at 1{sigma} around the heme, water, and PEG ligands. Possible hydrogen bonds with a cutoff of 3.2 Å are indicated by dashed lines. B, PEG molecule in the OxyC substrate-binding pocket. Heme and amino acid residues located closer than 4 Å from the PEG molecule are shown and labeled. C, stereo view comparing the distal (I-helix kink) and proximal sites (Cys ligand loop) of OxyB (light gray) and OxyC (dark gray). The figure was prepared using BOBSCRIPT (30, 33) and RASTER3D (31, 32).

 
In OxyC there is a water molecule (Wat-254) located in the groove of the I-helix. The water molecule forms hydrogen bonds with the carbonyl oxygen atoms of Ile-243 and Leu-244, respectively, and the amide of Glu-248. Thr-249 forms a hydrogen bond with the carbonyl oxygen atom of Gly-245. These interactions are typical for P450s, they interrupt the normal hydrogen bonding pattern of the backbone residues of the I-helix (27). This results in a kink of the I-helix that is believed to be important for oxygen binding and the proton shuttle mechanism. In OxyB the position of the conserved Thr is occupied by Asn-240 whose side chain points into the active site and forms a hydrogen bond with the Met-water molecule but not with the carbonyl oxygen of Ala-236 (7). The absence of the later interaction leads to a more open kink of the I-helix in OxyB and the appearance of an additional water binding site (Wat-39) in the groove of the I-helix; otherwise the other water binding pattern is similar in both proteins (Fig. 9C). The observation of a water molecule bound to the heme iron is consistent with the UV-visible spectra of OxyC indicating a low spin heme (see above); addition of PEG 550 monomethyl ether to the oxidized form causes no shift in the Soret band.

The iron is in the heme plane, the distance to the thiolate sulfur of Cys-356 is 2.41 Å. As in other P450s, the A propionic acid interacts in a bidentate fashion with Arg-298 (which interacts also with the carbonyl oxygen atom of Asn-296 and Wat246); the D propionic acid interacts with the imidazole nitrogen atoms of His-354 and His-105, respectively, and with Arg-109. The latter is solvent accessible and occupies the position of His-100, the residue that makes direct contact with the FMN-binding domain in the crystal structure of the complex of the heme and FMN domains of P450-BM3 (19).

The active site of OxyC is occupied by a PEG molecule (Fig. 9, A and B). It fits into a pocket formed by residues Phe-93, Gln-96, Ser-98, Thr-99, Ala-241, Leu-244, Gly-245, Asn-296, and the heme. The similar positions of the bound PEG molecule in both molecules in the asymmetric unit suggest that it might overlap with the substrate/product binding site of the enzyme and mimic the natural substrate/product. A similar observation was made in a study of the substrate-free peptide deformylase. It contained a PEG molecule in the substrate-binding pocket mimicking a natural substrate (28), which was confirmed later by solving the peptide deformylase structure in complex with its product, a Met-Ala-Ser tripeptide (29). However, trials to overlay the PEG molecule with the vancomycin aglycone were not successful. The pocket is large enough to accommodate the putative OxyC substrate, a bicyclic heptapeptide intermediate. As mentioned above, the timing of the OxyC reaction is unclear, and therefore the correct substrate is also unknown. The product might be vancomycin aglycone itself or a thioester derivative bound to a PCD. The natural substrate for OxyC is presently not available, and therefore it was not possible to obtain the protein-substrate complex. Because the product of OxyC might be the vancomycin aglycone, it was of interest to attempt to co-crystallize this molecule with OxyC. However, the crystal structure of OxyC obtained in the presence of vancomycin aglycone does not reveal any conformational changes in the protein, and there was no additional electron density in the substrate-binding pocket.

The timing of release of the putative linear heptapeptide from the peptidyl carrier domain of the vancomycin NRPS is presently unclear. It is possible that the heptapeptide must remain attached through its C terminus to a PCD of the NRPS in order to act as a substrate for all the oxy-proteins. There is a patch of conserved (among oxy-proteins, but not in all P450s) residues on the surface of OxyC (Fig. 7, red). It might be involved in binding of either the substrate, attached through its C terminus to a peptide carrier domain, or the peptide carrier protein itself.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1UED) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence may be addressed: Organisch-chemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland. Tel.: 41-1-635-4242; Fax: 41-1-635-6833. ** To whom correspondence may be addressed: Dept. of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstr. 29, 69120 Heidelberg, Germany. Tel.: 49-231-133-2738; Fax: 49-231-133-2797; E-mail: ilme.schlichting{at}mpimf-heidelberg.mpg.de.

1 The abbreviations used are: NRPS, nonribosomal peptide synthetase; PCD, peptide carrier domain; PEG, polyethylene glycol; r.m.s.d., root mean square difference; FMN, flavin mononucleotide. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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