The Second Enzyme in Pyrrolnitrin Biosynthetic Pathway Is Related to the Heme-Dependent Dioxygenase Superfamily

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.

The Ternary Complex of PrnB (the Second Enzyme in the Pyrrolnitrin Biosynthesis Pathway), Tryptophan, and Cyanide Yields New Mechanistic Insights into the Indolamine Dioxygenase Superfamily

“…Upon first inspection, the similarity to IDO is puzzling because PrnB does not add oxygen to the molecule but breaks the C2 N1 bond. The structural study proposed a series of chemical steps that are consistent with the initial steps of TDO mechanism, formation of the first carbon oxygen bond, but differ in the fate of the central intermediate, an alkylperoxy species (13). In a further difference from IDO, PrnB requires additional electrons during turnover.…”

“…Structural Biology-His-tagged PrnB Cys mutant (C21S, C60S, or C185S) was overproduced in P. fluorescence BL915 ⌬ORF1-4 (deletion of the gene cluster of the pyrrolnitrin biosynthesis pathway), purified, and crystallized as previously described (13,20) with two modifications; 7 M sterile filtered hemin was supplemented to the growth medium to ensure full incorporation of heme (21), and 0.1 M Bistris buffer (pH 6.2-6.4) was introduced to the crystallization condition and enhanced reproducibility of crystallization. The use of cyanide required appropriate safety considerations.…”

“…1a). The structure of PrnB (13) established that it is a member of the heme-b dioxygenase superfamily and closely related to indoleamine 2,3-dioxygenase (IDO) 2 and tryptophan 2, 3-dioxygenase (TDO) (14 -16). Both IDO and TDO react at indoleamine by breaking the C2 and C3 bond of the indole ring via insertion of molecular oxygen (Fig.…”

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

“…In addition to humans, TDO has also been found in other mammals, such as rats and mice, as well as in mosquitoes and bacteria (2, 5, 14 -18). Recently, a potential heme-dependent dioxygenase enzyme superfamily has been proposed (19). Moreover, several other heme-based proteins, such as myoglobin and hemoglobin, express dioxygenase activities from their mutant proteins under certain circumstances (20,21).…”

PhytopathogenicBurkholderiaSpecies in Bulb Plants in Bulgaria

“…Until now, there is no data about the synthesis by B. pyrrocinia of specific pigments, only antibiotics have been purified -bromo-derivatives of pyrrolnitrin, named bromonitrin A, B, and c (1,12). the pyrrolnitrin crystal is pale yellow in color but when expressed in Pseudomonas fluorescens, pyrrolnitrin B is red -variations due to its chemical and stereo-structure (9,31). Although such investigations were not subject to this study, the synthesis of such an antibiotic is a possible explanation.…”