Preliminary crystallographic characterization of PrnB, the second enzyme in the pyrrolnitrin biosynthetic pathway

Laurentis, Walter De; Leang, Khim; Hahn, Katrin; Podemski, Bianca; Adam, Ariane; Kroschwald, Sonja; Carter, Lester G.; Pée, Karl‐Heinz van; Naismith, James H.

doi:10.1107/s1744309106041649

Cited by 9 publications

(6 citation statements)

References 20 publications

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“…Complex Structure of PrnB with Cyanide-As described previously, we used the triple Cys mutant of PrnB (C21S, C60S, and C185S) because it is fully functional but, unlike native protein, does not aggregate (13,20). Crystals of native PrnB were soaked in 6 mM KCN to produce a binary complex structure of PrnB and cyanide.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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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

Zhu

Pée

Naismith

2010

Journal of Biological Chemistry

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Pyrrolnitrin (3-chloro-4-(2-nitro-3-chlorophenyl)pyrrole) is a broad-spectrum antifungal compound isolated from Pseudomonas pyrrocinia. Four enzymes (PrnA, PrnB, PrnC, and PrnD) are required for pyrrolnitrin biosynthesis from tryptophan. PrnB rearranges the indole ring of 7-Cl-L-tryptophan and eliminates the carboxylate group. PrnB shows robust activity in vivo, but in vitro activity for PrnB under defined conditions remains undetected. The structure of PrnB establishes that the enzyme belongs to the heme b-dependent indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) family. We report the cyanide complex of PrnB and two ternary complexes with both L-tryptophan or 7-Cl-L-tryptophan and cyanide. The latter two complexes are essentially identical and mimic the likely catalytic ternary complex that occurs during turnover. In the cyanide ternary complexes, a loop previously disordered becomes ordered, contributing to the binding of substrates. The conformations of the bound tryptophan substrates are changed from that seen previously in the binary complexes. In L-tryptophan ternary complex, the indole ring now adopts the same orientation as seen in the PrnB binary complexes with other tryptophan substrates. The amide and carboxylate group of the substrate are orientated in a new conformation. Tyr 321 and Ser 332 play a key role in binding these groups. The structures suggest that catalysis requires an L-configured substrate. Isothermal titration calorimetry data suggest D-tryptophan does not bind after cyanide (or oxygen) coordinates with the distal (or sixth) site of heme. This is the first ternary complex with a tryptophan substrate of a member of the tryptophan dioxygenase superfamily and has mechanistic implications.Pyrrolnitrin is a broad-spectrum potent antifungal compound (1) first isolated from Pseudomonas pyromania (2) and the active component of PYRO-ACE (treatment for fungal infections of skin). The gene cluster responsible for pyrrolnitrin biosynthesis was identified in Pseudomonas fluorescens (BL915) (3, 4) and subsequently in Pseudomonas pyrrocinia, Burkholderia cepacia LT4-12-W, Myxococcus fulvus Mx f147, and other pyrrolnitrin producing bacteria (5). Four conserved enzymes are involved in pyrrolnitrin biosynthesis and named PrnA, PrnB, PrnC, and PrnD, reflecting their order in catalysis. Introduction of the entire cluster to Escherichia coli results in the production of pyrrolnitrin, thereby demonstrating that the four genes are sufficient and essential for pyrrolnitrin biosynthesis in vivo (3). Genetic manipulation in P. fluorescens BL915 has identified the intermediate products from each enzyme in the pyrrolnitrin pathway, leading to the current model for the biosynthetic pathway (4). The first enzyme, PrnA (tryptophan 7-halogenase), incorporates the chlorine into the substrate tryptophan (6). Structural and biochemical analyses of both tryptophan 7 and 5-halogenase (7-10) have established a novel chemical mechanism of hypohalous acid formation at the flavin cofactor, followed by ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Zhu

Pée

Naismith

2010

Journal of Biological Chemistry

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“…Mass spectrometric analysis of the acetone extraction is consistent with the presence of heme b (peak at 615.95 nm; calculated 615.6 nm). Protein crystals were only obtained from the triple mutant protein and the details of the crystallization have been reported (15). The best results were obtained using with 1.5 μ l protein solution (16mg / ml 50mM Tris pH 7.5) plus 1.5 μ l of precipitant (0.22 M magnesium sulfate, 16 % w/v PEG 3350) irrespective of whether protein was incubated with tryptophan (as a saturated solution) or not.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2007

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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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“…174 The crystal structure of PrnB revealed this enzyme to be a member of the heme-dependent dioxygenase superfamily and suggested that a tryptophane peroxide is the primary oxidation product. 175,176 The Rieske N-oxygenase PrnD, which generates the nitro group, was biochemically characterized, [177][178][179] and hydroxylamine and nitroso analogs were identified as intermediates. 179 PrnF was established as an NADH-dependent reductase of PrnD.…”

Section: Plantsmentioning

confidence: 99%

Metabolites from symbiotic bacteria

2009

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Preliminary crystallographic characterization of PrnB, the second enzyme in the pyrrolnitrin biosynthetic pathway

Cited by 9 publications

References 20 publications

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

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

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

Metabolites from symbiotic bacteria

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