View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Article Ralfuranone Biosynthesis in Ralstonia solanacearum Suggests Functional Divergence in the Quinone Synthetase Family of Enzymes Barbara Wackler,1 Patrick Schneider,1 Jonathan M. Jacobs,2 Julia Pauly,1 Caitilyn Allen,2 Markus Nett,3 and Dirk Hoffmeister1,* 1Department Pharmaceutical Biology at the Hans-Kno¨ ll-Institute, Friedrich-Schiller-Universita¨ t Jena, Beutenbergstrasse 11a, 07745 Jena, Germany 2Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706, USA 3Leibniz Institute for Natural Product Research and Infection Biology—Hans-Kno¨ ll-Institute, Beutenbergstrasse 11a, 07745 Jena, Germany *Correspondence: [email protected] DOI 10.1016/j.chembiol.2011.01.010 SUMMARY VsrAD and PhcA, and secondary metabolism has been demon- strated and led to the identification of a new small molecule, Ralstonia solanacearum is a destructive crop plant ralfuranone (4-phenylfuran-2(5H)-one, 1, Figure 1)(Schneider pathogen and produces ralfuranone, i.e., a mono- et al., 2009). Although achiral and little functionalized, this bicy- phenyl-substituted furanone. Extensive feeding clic secondary product attracted our attention as the origin of experiments with 13C-labeled L-phenylalanine now the carbon atoms was elusive. Initially, we hypothesized that proved that all carbon atoms of the heterocycle the aromatic system may be derived from L-phenylalanine, and derive, after deamination, from this aromatic amino that transfer of malonyl-CoA to the former may provide the carbon atoms to complete furanone synthesis. Chemical char- acid. A genetic locus was identified which encodes acterization of a new, yet related natural product, ralfuranone B the aminotransferase RalD and the furanone synthe- (2, Figure 1), prompted us to propose a revised biosynthetic tase RalA. The latter is a tridomain nonribosomal mechanism. Support came from feeding experiments with 13C- peptide synthetase (NRPS)-like enzyme which was labeled L-phenylalanine and from the genetic and biochemical characterized (1) biochemically by the ATP-pyro- characterization of two biosynthesis enzymes, the aminotrans- phosphate exchange assay, and (2) genetically ferase RalD and the furanone synthetase RalA. The latter is a tri- through gene inactivation and transcriptional anal- domain protein reminiscent of fungal quinone synthetases and ysis in axenic culture and in planta. This is the first shares an identical domain architecture and a high degree of study to our knowledge on the biochemical and similarity with AtrA from the basidiomycete Tapinella panuoides genetic basis of R. solanacearum secondary meta- (Schneider et al., 2008). However, our data on RalA imply a mech- bolism. It implies new chemistry for NRPSs, as anism for furanone biosynthesis, which is dissimilar from quinone synthetase. RalA-mediated biosynthesis requires C-C-bond and subsequent C-O-bond formation to establish the fur- RESULTS AND DISCUSSION anone ring system. Feeding Experiments 13 INTRODUCTION Stable isotope feeding was initiated with 1 mM [1,2- C2] acetate as carbon source to probe its metabolic fate in the biosynthesis The Gram-negative bacterium Ralstonia solanacearum is a noto- of 1. Unexpectedly, R. solanacearum failed to produce 1 under rious plant pathogen which invades its hosts in the rhizosphere these conditions. Therefore, we fed 1 mM [2-13C] pyruvate to and colonizes their xylem vessels, eventually leading to wilt the bacterium, as genomic data supported an intracellular disease (Hayward, 1991). Its epithet implies a dependence on conversion into acetyl-CoA via the pyruvate dehydrogenase solanaceaous plants, and, in fact, R. solanacearum threatens multienzyme complex. Albeit production of 1 could be restored the health of agriculturally and commercially relevant crop plants in this way, no 13C enrichment was detected at C-2 (or any other within this family, such as tomato and potato. However, this carbon atom) disproving our initial assumption that acetate bacterium also parasitizes numerous species of other mono- serves as a building block. Concurrently, 2 was isolated from and dicotyledonous plant families including species of impor- the culture broth of R. solanacearum. Its chemical structure is tance for staple food production, such as banana (Hayward, new, yet strongly resembling the fungal gymnoascolides (Fig- 1991). Given its significance as a crop plant pathogen, it is ure 1)(Clark et al., 2005). A related compound, 3-carboxy-2,4-di- surprising that data on its secondary metabolome have only phenyl-but-2-enoic anhydride (Figure 1), was also described as begun to emerge. One report is pertaining to biosynthesis and a fungal metabolite (Hamasaki and Nakajima, 1983). Combined regulation of 3-hydroxy-oxindole (Delaspre et al., 2007). More with the results of the feeding experiment, the structure of 2 recently, crosstalk between the global virulence regulators made us reconsider our working hypothesis on the origin of the 354 Chemistry & Biology 18, 354–360, March 25, 2011 ª2011 Elsevier Ltd All rights reserved Chemistry & Biology Ralfuranone Biosynthesis 14 Figure 1. Chemical Structures of Ralstonia Metabolites Ralfuranone (1) and Ralfura- 12 none B (2), and Related Natural Products O O O 10 of Other Sources 2 2 O HO O 4 O O O O 6 4 6 5 5 O O OH 9 7 1 9 7 2 8 8 3-Carboxy-2,4-diphenyl- Gymnoascolide A Xenofuranone B but-2-enoic anhydride (Gymnoascus reessii) (Xenorhabdus szentirmaii) 2007, 2008). Their genes are located (Aspergillus sp.) in loci which include aromatic amino- transferase genes (tdiD and atrD, respec- tively). A putative aminotransferase gene, 1 furanone carbons. The broad-band 1H-decoupled 13C NMR RSp1424/ectB, coding for a 434 aa enzyme and annotated as di- 13 spectrum of 1 labeled from a feeding experiment with [U- C9] aminobutyrate:2-oxoglutarate transaminase gene, was present L-phenylalanine indicated extensive enrichment and coupling in the vicinity of RSp1419 as well (Figure 2). Consequently, these throughout the molecule (see Figure S1 available online). For two genes, hereafter referred to as ralA and ralD (GenBank quantitative purposes, the absolute 13C enrichment of the meth- accession: HQ864831 and HQ864832, respectively) were inacti- ylene carbon atom at C-5 was determined by integration of the vated. The aminotransferase RalD was not strictly required for methylene proton signal and its 13C satellites in the 1H NMR ralfuranone synthesis, as production was still active in a ralD spectrum. The integral of the corresponding 13C signal in the disruption mutant, albeit at a reduced level (Figure 3). A very inverse gated 1H-decoupled 13C NMR spectrum was then similar effect was found with terrequinone biosynthesis upon used as the reference against which enrichments at other carbon aminotransferase tdiD gene knockout, where production was atoms were calculated (Table 1). First order multiplet analysis in impaired, but not abolished, possibly due to a partial comple- the 13C NMR spectrum suggested an assembly of 1 from two mentation by housekeeping aminotransferases (Schneider discrete phenylalanine-derived fragments, that is, a C6-C2 et al., 2007). Mutants in which ralA was disrupted by a kanamycin moiety covering the carbon atoms C-4 to C-9 of ralfuranone resistance cassette completely lost the capacity to produce and a C2 unit composed of the remaining carbon atoms C-2 ralfuranone (Figure 3). Therefore, we conclude that RalA plays and C-3, respectively. This was further corroborated by consis- an essential role for ralfuranone biosynthesis. Although the 13 tent C-enrichments within the C6-C2 versus C2 building block ecological role of ralfuranone biosynthesis has not been clarified (Table 1). As both fragments exhibit high enrichment levels, we yet, a genetic screen found that R. solanacearum expresses ralA assume that they derive intact without extensive modification in planta (Brown and Allen, 2004). Gene expression analysis with from the fed precursor. Moreover, it appears plausible that 2 DNA microarrays also identified that both ralA and ralD are highly precedes the formation of 1, and that a debenzylation reaction expressed during wilt disease of tomato and in rich medium could give rise to the latter. C-2 and C-3 would thus originate (Table 2)(Jacobs et al., 2010). The ralA gene is embedded in from the carboxyl and the a-carbon in L-phenylalanine, respec- an apparently larger locus of putative natural product genes tively. Consistent with this assumption, administration of [3-13C] that implies more complex metabolites than just ralfuranones. L-phenylalanine (230 mM final) resulted in the exclusive incorpo- However, these products have not been detected yet. In partic- ration of label at C-4. ular, the presence of RSp1422, located between ralA and ralD (Figure 2) and encoding a monomodular NRPS is incompatible Ralfuranone Biosynthesis Genes with the structure of 1 and 2, respectively, as neither has Our results demonstrate that all carbon atoms of 1 are derived a peptide bond. Notably, RSp1422 is moderately and slightly from L-phenylalanine and imply that a potential genetic locus expressed in rich medium and in planta, respectively. This encodes an enzyme to activate L-phenylalanine or, in conjunc- tion with aminotransferase activity, phenylpyruvate. A multigene Table 1. 13C Abundance and Coupling Constants in Ralfuranone locus which meets these requirements was