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. 2007 Oct 30;46(43):12393-404.
doi: 10.1021/bi7012189. Epub 2007 Oct 9.

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily

Affiliations

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily

Walter De Laurentis et al. Biochemistry. .

Erratum in

  • Biochemistry. 2007 Dec 18;46(50):14733. Johnson, Kenneth A [added]

Abstract

Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.

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Figures

Figure 1
Figure 1
A stereo ribbon representation of the structure of PrnB, the core helical domain is colored cyan and the N-terminal capping domain is colored blue. The cyan colored helices are found in TDO, the N-terminal domain is not. The disordered loop is marked by a * at each end. The side chains of three cysteine residues (20, 60 and 175) which were mutated to serine are shown in sticks. The heme group and the proximal H313 ligand are shown in sticks with carbon yellow, nitrogen blue and oxygen red.
Figure 2
Figure 2
(a) The complexes of PrnB with substrates 7-Cl-L-tryptophan, 7-Cl-D-tryptophan and L-tryptophan (D-tryptophan overlaps with 7-Cl-D-tryptophan and is not shown). The protein is colored as Figure 1, non carbon atoms are colored as Figure 1, chlorine atoms are purple, for 7-Cl-L-tryptophan carbon atoms are colored yellow, carbons in 7-Cl-D-tryptophan are colored orange and carbons in L-tryptophan are colored salmon. Although the chloro indole rings of 7-Cl-L-tryptophan and 7-Cl-D-tryptophan overlap, the main chain atoms do not. L-tryptophan binds in a different manner to the other complexes, although the part of the indole rings overlap. The proximal ligand to the heme (H313) is shown. In 7-Cl-L-tryptophan and L-tryptophan, the amino nitrogen atom ligates to the iron, whereas in 7-Cl-D-tryptophan one of the carboxyl oxygen atoms ligates to the iron. (b) The active site of PrnB. The residues that surround the 7-Cl-L-tryptophan ligand are shown. The oxyanion hole at A224 is labeled and the hydrogen bonds shown. Atoms are colored as Figure 2a. (c) The subtle differences in the active sites of PrnB (colored as Figure 2a) and TDO(carbon atoms green, other atoms colored as Figure 2a). The superposition is based on the heme groups of each protein, the different orientation of the (d) Superposition of the L-tryptophan complexes of PrnB and TDO (PDB code 2NW8); atoms colored as Figure 2c. The binding sites are mutually exclusive, F201 in PrnB prevents L-tryptophan binding as seen in TDO, whilst H55 of TDO prevents any of the binding modes for tryptophan seen in PrnB. (e) The substrate complexes of PrnB and TDO show that despite quite different orientations the CD2 atom (marked with *) where the peroxide is predicted to be inserted are similarly positioned relative to the heme.
Figure 2
Figure 2
(a) The complexes of PrnB with substrates 7-Cl-L-tryptophan, 7-Cl-D-tryptophan and L-tryptophan (D-tryptophan overlaps with 7-Cl-D-tryptophan and is not shown). The protein is colored as Figure 1, non carbon atoms are colored as Figure 1, chlorine atoms are purple, for 7-Cl-L-tryptophan carbon atoms are colored yellow, carbons in 7-Cl-D-tryptophan are colored orange and carbons in L-tryptophan are colored salmon. Although the chloro indole rings of 7-Cl-L-tryptophan and 7-Cl-D-tryptophan overlap, the main chain atoms do not. L-tryptophan binds in a different manner to the other complexes, although the part of the indole rings overlap. The proximal ligand to the heme (H313) is shown. In 7-Cl-L-tryptophan and L-tryptophan, the amino nitrogen atom ligates to the iron, whereas in 7-Cl-D-tryptophan one of the carboxyl oxygen atoms ligates to the iron. (b) The active site of PrnB. The residues that surround the 7-Cl-L-tryptophan ligand are shown. The oxyanion hole at A224 is labeled and the hydrogen bonds shown. Atoms are colored as Figure 2a. (c) The subtle differences in the active sites of PrnB (colored as Figure 2a) and TDO(carbon atoms green, other atoms colored as Figure 2a). The superposition is based on the heme groups of each protein, the different orientation of the (d) Superposition of the L-tryptophan complexes of PrnB and TDO (PDB code 2NW8); atoms colored as Figure 2c. The binding sites are mutually exclusive, F201 in PrnB prevents L-tryptophan binding as seen in TDO, whilst H55 of TDO prevents any of the binding modes for tryptophan seen in PrnB. (e) The substrate complexes of PrnB and TDO show that despite quite different orientations the CD2 atom (marked with *) where the peroxide is predicted to be inserted are similarly positioned relative to the heme.
Figure 3
Figure 3
(a) HPLC-analysis of the in vitro activity assay of PrnB incubated with racemic 7-chlorotryptophan. The bottom line shows monodechloroaminopyrrolnitrin (MCAP) the product formed by PrnB from 7-chlorotryptophan as a standard. The other lines show from bottom to top increasing volume of crude extracts. The numbers on the right hand side give the amount of MCAP corresponding to the peak. (b) HPLC-analysis of the in vitro activity assay of PrnB incubated with racemic 7-chlorotryptophan and the pure D- and L-enantiomers, respectively. The lines from bottom to top show: MCAP standard; incubation with racemic 7-chlorotryptophan (0.5 mM and 0.25 mM final concentration, respectively); incubation with 0.5 mM 7-chloro-D-tryptophan; incubation with 0.5 mM 7-Cl-L-tryptophan. (c) HPLC-analysis of in vitro activity assays of PrnB. The lines from bottom to top show: MCAP as the standard; crude extract of the host strain P.fluorescens BL915 Δorf1-4; dialyzed PrnB-containing extract; cell-free crude extract containing PrnB; restoration of PrnB activity by incubation of dialyzed crude extract together with non-dialyzed crude extract of the host strain.
Figure 4
Figure 4
(a) OTTLE potentiometry of PrnB in the absence of L-Tryptophan (circles), + 20 mM L-Tryptophan (triangles pointing up), + 20 mM D-Tryptophan (triangles pointing down) and 10 mM 7-Chloro-L-Tryptophan. In each case the filled and empty shapes correspond to the reductive and oxidative traces respectively. Each data set is fit using the Nernst equation and the fit is coloured according to the corresponding label. (b) Decay of oxyferrous PrnB. Upper Panel. Spectral changes associated with the decay of the oxyferrous PrnB species. Lower Panel. Exponential decrease in absorbance at 543 nm used to determine the rate of oxyferrous PrnB decay. Red line indicates single exponential
Figure 5
Figure 5
(a) (a) Perpendicular mode X-band EPR spectra of native ferric PrnB in the absence of tryptophan (black line), ferric PrnB + 20 mM L-tryptophan (red line) and ferric PrnB + 20 mM D-tryptophan (blue line). Conditions: microwave frequency, 9.67 GHz; microwave power, 2 mW; modulation amplitude, 10 G; temperature, 10 K; scan speeds and time constants are the same for all spectra. Spectra have been adjusted for differences in the enzyme concentration and receiver gain where required. Gains were typically of the order of 2 × 105 - 8 × 105. (b) Room-temperature MCD at 5 T in the UV/visible region of native ferric PrnB in the absence of tryptophan (black line), ferric PrnB + 20 mM L-tryptophan (red line) and ferric PrnB + 20 mM D-tryptophan (blue line). All spectra were recorded at pH 7.0. (c) Room-temperature MCD at 5 T in the near-IR region of native ferric PrnB in the absence of tryptophan (black line), ferric PrnB + 20 mM L-tryptophan (red line) and ferric PrnB + 20 mM D-tryptophan (blue line). All spectra were recorded at pH 7.0 with 50 μM enzyme.
Scheme 1
Scheme 1
Pyrrolnitrin pathway. The reaction catalyzed by PrnB is boxed.
Scheme 2
Scheme 2
The reaction catalyzed by TDO and by IDO.
Scheme 3
Scheme 3
The three fused ring intermediate proposed to form during PrnB catalysis. This has analogues in the natural product pyrroindomicyn (26) and biomimetic chemistry (pyrrolobenzoxazine core) (25), an arylpyrrol structure has been found in pyrrolnitrin producing bacteria (28, 29).
Scheme 4
Scheme 4
The proposed chemical route followed during PrnB catalysis, the tricyclic ring structure is boxed. A route to the pyrroindomycin core and ayrlpyrrole core structures are shown (24).

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