The structure of dimethylallyl tryptophan synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria Ute Metzgera,1, Christoph Schallb,1, Georg Zocherb, Inge Unso¨ lda, Edyta Stecc, Shu-Ming Lic, Lutz Heidea,2, and Thilo Stehleb,d aPharmazeutisches Institut, Universita¨t Tu¨ bingen, 72076 Tu¨bingen, Germany; bInterfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨ bingen, 72076 Tu¨bingen, Germany; cInstitut fu¨r Pharmazeutische Biologie, Universita¨t Marburg, 35037 Marburg, Germany; and dDepartment of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232 Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved July 9, 2009 (received for review May 5, 2009) Ergot alkaloids are toxins and important pharmaceuticals that are farnesyl diphosphate synthase (11, 12), DMATS does not require produced biotechnologically on an industrial scale. The first com- magnesium or other divalent cations for its enzymatic activity, mitted step of ergot alkaloid biosynthesis is catalyzed by dimethy- although addition of 4 mM CaCl2 moderately increases its reaction lallyl tryptophan synthase (DMATS; EC 2.5.1.34). Orthologs of velocity (10). The structural gene coding for DMATS in Claviceps, DMATS are found in many fungal genomes. We report here the termed dmaW, was identified by Tsai et al. (13). A similar gene, x-ray structure of DMATS, determined at a resolution of 1.76 Å. A fgaPT2, exists in the biosynthetic gene cluster of fumigaclavine, in complex of DMATS from Aspergillus fumigatus with its aromatic the genome sequence of A. fumigatus. Expression of the DMATS substrate L-tryptophan and with an analogue of its isoprenoid sequence from A. fumigatus (hereafter referred to as FgaPT2) as a substrate dimethylallyl diphosphate reveals the structural basis of his-tagged protein yielded the initial enzyme of ergot alkaloid this enzyme-catalyzed Friedel-Crafts reaction, which shows strict biosynthesis. This enzyme was characterized in homogeneous form regiospecificity for position 4 of the indole nucleus of tryptophan after heterologous expression (14). A very similar enzyme was later -as well as unusual independence of the presence of Mg2؉ ions. The described from the fungus Malbranchea aurantiaca (15), and or BIOCHEMISTRY 3D structure of DMATS belongs to a rare ␤/␣ barrel fold, called thologs of DMATS have been found in many other fungal genomes. prenyltransferase barrel, that was recently discovered in a small Recently, several enzymes with sequence similarity to DMATS group of bacterial enzymes with no sequence similarity to DMATS. have been cloned from fungal genomes and were expressed, These bacterial enzymes catalyze the prenylation of aromatic purified, and biochemically characterized, as reviewed by Steffan et substrates in the biosynthesis of secondary metabolites (i.e., a al. (16). These enzymes show different substrate specificities, reaction similar to that of DMATS). catalyzing the prenylation of tryptophan or derivatives thereof. In addition, they exhibit different regiospecificities because they can EC 2.5.1.34 ͉ ergot alkaloids ͉ PT barrel ͉ ABBA prenyltransferase attach the prenyl group to different carbons or to the nitrogen atom of the indole nucleus. Furthermore, the prenyl group can be he ergot alkaloids are among the most important natural attached via its C-1 (‘‘regular prenylation’’) or its C-3 (‘‘reverse Tpharmaceuticals and toxins in human history (1). Ergots are prenylation’’). resting structures (sclerotia) of certain fungi that parasitize ears of The fungal indole PTs are key enzymes in the biosynthesis of a grain. The medicinal uses of ergots may date back to 600 B.C. in fascinating array of structurally diverse prenylated indole alkaloids Mesopotamia (now Iraq) (2) and were certainly well described in in fungi (17). Currently, BLAST searches reveal more than 100 the 16th century (3). Both the natural ergot alkaloids and their entries with sequence similarity to DMATS in the database. The semisynthetic derivatives are in widespread use in modern medicine fungal indole PTs that have been biochemically investigated show and exhibit a broad spectrum of pharmacological activities, includ- remarkable flexibility for their aromatic substrates, and these ing uterotonic activity, modulation of blood pressure, control of the enzymes may be of considerable interest for chemoenzymatic secretion of pituitary hormones, migraine prevention, and dopa- synthesis of bioactive compounds (16). minergic and neuroleptic activities (4). Ingestion of ergot-infected Although the crystallization of DMATS from Claviceps was grass or grains caused severe epidemics in the Middle Ages in reported in 1981 (18), no structure of an indole PT has been Europe, both in humans and in animals, and ergot-infested grasses determined. We now report the crystallization and x-ray structural continue to produce serious epidemics in livestock today (1, 5). The analysis of FgaPT2, the enzyme catalyzing the first committed step fame of ergot alkaloids is also attributable to the potent halluci- of ergot alkaloid biosynthesis in A. fumigatus. Unexpectedly, the nogenic activity of lysergic acid diethylamide, originally reported by structure of this enzyme was found to belong to a very rare ß/␣ Stoll and Hofmann (6). barrel fold, called PT fold (19). This fold has only recently been The ergot alkaloids, which are characterized by the ergoline ring system (Fig. 1), can be divided into 2 major classes: the amides of lysergic acid, which are produced by plant-associated Author contributions: S.-M.L., L.H., and T.S. designed research; U.M., C.S., G.Z., I.U., and E.S. performed research; U.M., C.S., G.Z., L.H., and T.S. analyzed data; and U.M., C.S., G.Z., L.H., fungi of the family Clavicipitaceae, and the clavine alkaloids, and T.S. wrote the paper. which are primarily produced by members of the fungal order The authors declare no conflict of interest. Eurotiales such as Aspergillus fumigatus, currently the most This article is a PNAS Direct Submission. common agent of invasive mycosis in humans (7). Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, The first committed step in ergot alkaloid biosynthesis is the www.rcsb.org (PDB ID codes 3I4X and 3I4Z). prenylation of L-tryptophan, catalyzed by the 4-dimethylallyl tryp- 1U.M. and C.S. contributed equally to this work. tophan synthase (DMATS; E.C. 2.5.1.34) (Fig. 1). The enzyme was 2To whom correspondence should be addressed at: Pharmazeutische Biologie, Pharma- initially described from Claviceps in 1971 (8) and, later on, was zeutisches Institut, Universita¨tTu¨ bingen, Auf der Morgenstelle 8, 72076 Tu¨bingen, Germany. purified to apparent homogeneity (9, 10). It is a soluble ho- E-mail: [email protected]. Ϸ modimeric protein with an apparent molecular weight of 105 This article contains supporting information online at www.pnas.org/cgi/content/full/ kDa. In sharp contrast to other prenyltransferases (PTs) such as 0904897106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904897106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 4' DMAPP 3CH CH3 CH NH2 3 NH2 α 3' 2' β COOH COOH 3CH O P P PPi 4 3'' 1' 3 5 2 6 N 1 DMATS N 7 (FgaPT2) H H L-tryptophan dimethylallyl tryptophan (DMAT) RO C CH3 AcO D H H N N N H CH3 CH3 A C H B N N N H H H R ergoline ring system R = OH lysergic acid R = H fumigaclavine A OH 3 OCH R = N ergotamine NH CH3 N O O H R = CCHfumigaclavine C CH3 CH2 C2H5 lysergic acid R = N diethylamide C2H5 (LSD) Fig. 1. Reaction catalyzed by DMATS, and structures of ergot alkaloids. discovered in a group of bacterial enzymes that also catalyze the search (22) for structurally related proteins revealed the bacterial prenylation of aromatic substrates but do not show detectable enzyme NphB (formerly called Orf2) as the only significant match primary sequence similarity to the fungal indole PTs (20, 21). (z ϭ 16.5). NphB is involved in the biosynthesis of the secondary metabolite naphterpin (19) and, remarkably, catalyzes an aromatic Results and Discussion prenylation reaction similar to that of FgaPT2. The comparison of Determination of the Structure of the DMATS FgaPT2. The crystal the structures of FgaPT2 and NphB (Fig. S1) clearly demonstrates structure of unliganded FgaPT2 was determined at 1.76 Å resolu- that both proteins share a common architecture, which has been tion with the use of single-wavelength anomalous dispersion and called PT barrel in the case of NphB (19). However, with 459 noncrystallographic symmetry averaging [supporting information residues, FgaPT2 is significantly larger than the 307-residue– (SI), Table S1]. A ternary complex containing both substrates was containing NphB. The additional residues account for extended prepared by incubating an FgaPT2 crystal for 15 min at 4 °C with loop regions and extra ␣-helices in FgaPT2 (Fig. S1). 3 mM L-tryptophan and with 2 mM dimethylallyl S-thiolodiphos- The FgaPT2 and NphB structures both contain 5 structurally phate (DMSPP). DMSPP serves as an analogue to the natural similar repeating units, which can be described as ␣␣␤␤ repeats substrate, dimethylallyl diphosphate (DMAPP). The structure of (Fig. S1). The 2 ␣-helices pack tightly against the 2 ␤-strand units the ternary complex was then determined to a resolution of 2.2 Å in each repeat, forming a hydrophobic core. Superposition shows by molecular replacement using rigid body refinement of the that the core regions of the 5 repeats agree reasonably well with unliganded structure. Initial difference electron density maps each other, mostly at positions that contribute to the hydrophobic clearly revealed the presence of both ligands in the complex core in each repeat, whereas the connecting regions exhibit signif- structure and also showed that a number of side chains in the active ␣ site had rearranged in response to the presence of the ligands. icant diversity. FgaPT2 has an additional N-terminal -helix, Crystallographic refinement of both structures yielded models that whereas NphB shows an extra C-terminal helix (Fig.