structural communications Acta Crystallographica Section F Structural Biology Structure of an Aspergillus fumigatus old yellow Communications enzyme (EasA) involved in ergot alkaloid ISSN 2053-230X biosynthesis Annemarie S. Chilton, Ashley L. The Aspergillus fumigatus old yellow enzyme (OYE) EasA reduces chano- Ellis and Audrey L. Lamb* clavine-I aldehyde to dihydrochanoclavine aldehyde and works in conjunction with festuclavine synthase at the branchpoint for ergot alkaloid pathways. The ˚ Molecular Biosciences, University of Kansas, crystal structure of the FMN-loaded EasA was determined to 1.8 A resolution. 1200 Sunnyside Avenue, Lawrence, KS 66045, The active-site amino acids of OYE are conserved, supporting a similar USA mechanism for reduction of the / -unsaturated aldehyde. The C-terminal tail of one monomer packs into the active site of a monomer in the next asymmetric unit, which is most likely to be a crystallization artifact and not a mechanism of Correspondence e-mail: [email protected] self-regulation. Received 22 July 2014 1. Introduction Accepted 21 August 2014 Ergot alkaloids are produced by fungi and plants and have histori- cally been used medicinally and for psychedelic recreation (lysergic acid derivatives), and have been the cause of poisonings with symp- PDB reference: EasA, 4qnw toms that were attributed to witchcraft (Schardl et al., 2006). These secondary metabolites are derived from tryptophan and dimethylallyl diphosphate (DMAPP) and are characterized by the tetracyclic ergoline ring system, of which festuclavine is one of the simplest examples, or characterized by the tricyclic ring system exemplified by chanoclavine-I aldehyde (Fig. 1a; Schardl et al., 2006). Isotope- feeding studies suggest that chanoclavine-I aldehyde may be a common intermediate for the biosynthesis of all ergot alkaloids (Floss et al., 1974). Conversion of chanoclavine-I aldehyde to dihy- drochanoclavine aldehyde by Aspergillus fumigatus has been reported to require an old yellow enzyme (OYE) called EasA (Cheng et al., 2010a,b; Coyle et al., 2010; Geu-Flores et al., 2012) or FgaOx3 (Wallwey et al., 2010) for the reduction of the / -unsaturated alde- hyde. The dihydrochanoclavine aldehyde is subsequently cyclized to form an iminium compound. It is unknown whether the cyclization is enzyme-catalyzed. A second reduction step to produce festuclavine from the iminium compound is performed by the NADH-dependent festuclavine synthase FgaFS (Wallwey et al., 2010). The Saccharomyces pastorianus old yellow enzyme was the first flavoenzyme to be identified (Warburg & Christian, 1932). Members of the OYE family are found in bacteria, fungi and plants, and catalyze the reduction of activated carbon–carbon double bonds (for excellent reviews, see Stuermer et al., 2007; Williams & Bruce, 2002). Here, we present the structural characterization of the A. fumigatus OYE EasA involved in ergot alkaloid biosynthesis. These data allow us to corroborate the proposed mechanism and to provide insight into the oligomerization of EasA in solution and in the crystal. 2. Materials and methods 2.1. Macromolecule production The preparation of the expression vector has been described previously (Cheng et al., 2010a). The expression vector, which generates C-terminally histidine-tagged EasA, was transformed into Escherichia coli BL21(DE3) cells, which were grown in LB broth containing 50 mgmlÀ1 kanamycin at 37C with shaking (175 rev minÀ1) for 2.5 h. The temperature was reduced to 15C for # 2014 International Union of Crystallography 1 h until an optical density at 600 nm of 0.1 was reached. Protein All rights reserved expression was induced with 50 mM IPTG and the cultures were 1328 doi:10.1107/S2053230X14018962 Acta Cryst. (2014). F70, 1328–1332 structural communications incubated for 64 h. Cells were harvested by centrifugation (3800g, (GE Healthcare) pre-equilibrated with 50 mM Tris–HCl, 150 mM 4C, 10 min). The cell pellet was resuspended in 10 ml 5 mM imida- sodium citrate, 10% glycerol pH 8.0. The fractions containing EasA zole, 300 mM NaCl, 20 mM Tris–HCl pH 8.0, 10% glycerol (buffer A). were pooled, concentrated to 3 mg mlÀ1 using an Amicon stirred cell The cells were disrupted using a French pressure cell (241 MPa) and with a YM-30 membrane and stored at À80C. Approximately 9 mg cellular debris was removed by centrifugation (20 800g,4C, 1 h). The EasA was purified from 4 l culture. EasA eluted from the size- lysate was applied onto 50 ml Ni–NTA resin (Qiagen) in a Kontes exclusion column was 85% FMN-bound. The amount of FMN column equilibrated with buffer A. The column was shaken to mix the loading was determined by comparing the concentration of EasA, lysate with the resin and incubated overnight at 4 C on a gently tilting determined by measuring the A280 (in deionized water " = base. After 21 h, EasA was eluted from the Ni–NTA column with a 56 840 MÀ1 cmÀ1), and the concentration of FMN, determined by À1 À1 step gradient of imidazole (5, 25, 50, 100, 150 and 300 mM) in buffer measuring the A446 (" = 11 500 M cm ). A. EasA primarily eluted at 150 mM imidazole. To load the EasA protein with FMN (flavin mononucleotide), a 2.2. Crystallization previously published procedure (Gatti et al., 1994) was modified. A Crystals were grown at 25C using the hanging-drop vapor-diffu- Red Sepharose (GE Healthcare) column was pre-equilibrated with sion method with 750 ml reservoir solution consisting of 230 mM three column volumes of buffer B (50 mM potassium phosphate ammonium sulfate, 100 mM MES pH 6.0, 30% PEG 8000. The monobasic pH 7.0, 10% glycerol). The 150 mM imidazole Ni–NTA hanging drops consisted of 1.5 ml reservoir solution and 1.5 ml fractions (50 ml) were concentrated to 25 ml and diluted in buffer B 24 mg mlÀ1 protein. Yellow crystals were fully developed in 4 d with to a final estimated salt concentration of 25 mM NaCl. The sample approximate dimensions of 160 Â 40 Â 20 mm. For data collection, was injected and washed with eight column volumes of buffer B crystals were transferred to the reservoir solution supplemented with followed by a linear gradient of 0–600 mM NaCl (five column 20% glycerol solution and were flash-cooled by plunging them into volumes) in buffer B to wash out unbound sample. The column was liquid nitrogen. washed with two column volumes of buffer B with 10 mM FMN. EasA was eluted using a 20-column-volume linear gradient from 0 to 2 M NaCl with 10 mM FMN in buffer B. 2.3. Data collection and processing The fractions containing EasA from the Red Sepharose column X-ray diffraction data were collected on beamline 11-1 at the were pooled and applied onto a Superdex 200 size-exclusion column Stanford Synchrotron Radiation Laboratory (SSRL), Stanford, Figure 1 EasA mechanism and overall structure. (a) The reaction catalyzed by the old yellow enzyme EasA from A. fumigatus, the conversion of chanoclavine-I aldehyde to dihydrochanoclavine, is labeled in red. The next two reactions are hypothesized to be spontaneous. The final reaction for the generation of festuclavine is catalyzed by FgaFS, festuclavine synthase. (b) EasA is a classic / -barrel, shown as a blue cartoon. The FMN (green sticks) is bound in the active-site funnel composed by eight parallel strands of the -barrel. The backside of the barrel is closed by a two-stranded -sheet: the two strands that precede the first strand of the barrel at the N-terminus of the protein. (c) The C-terminal tail of the red monomer packs in the active site of the blue monomer in the next asymmetric unit. The location of the active site is noted by the inclusion of the FMN as green sticks. Protein structure pictures were generated in PyMOL (Schro¨dinger). Acta Cryst. (2014). F70, 1328–1332 Chilton et al. EasA 1329 structural communications Table 1 Table 2 Data collection and processing. Structure refinement. Values in parentheses are for the outer shell. Values in parentheses are for the outer shell. Diffraction source SSRL beamline 11-1 Resolution range (A˚ ) 35.630–1.799 (1.846–1.799) Wavelength (A˚ ) 0.97945 No. of reflections 40082 (2851) Temperature ( C) À173 Final Rcryst 0.178 (0.221) Detector PILATUS 6M Final Rfree 0.221 (0.273) Crystal-to-detector distance (mm) 260 Total unique observations 40082 Rotation range per image ()0.5No. of non-H atoms Total rotation range () 180 Protein 2916 Exposure time per image (s) 20 Solvent (sulfate and glycerol) 26 Space group P212121 FMN 31 a, b, c (A˚ ) 62.5, 65.9, 115.2 Water 239 , , () 90, 90, 90 R.m.s. deviations Resolution range (A˚ ) 38.41–1.80 (1.90–1.80) Bonds (A˚ ) 0.026 Total No. of reflections 162475 (23165) Angles () 2.02 No. of unique reflections 44595 (6332) Overall mean B factor (A˚ 2) 21.6 Completeness (%) 99.3 (97.6) Multiplicity 3.6 (3.7) hI/(I)i 23.6 (3.3) percentage of FMN bound (0–6%) by a comparable purification R 0.040 (0.399) merge strategy using nickel-affinity chromatography (the protein is histi- dine-tagged). However, a purification step was added in which Red Califonia, USA using a wavelength of 0.97945 A˚ . Data (180) were Sepharose is used to bind EasA (owing to its affinity for the flavin) collected as 0.5 oscillation images (20 s exposure per frame) using and the protein is eluted from the column using sodium chloride in Blu-Ice (Soltis et al., 2008) with a crystal-to-detector distance of the presence of FMN. This is an adaptation of the protocol used to 260 mm at À173C. The crystals were assigned to space group exchange the natural flavin in p-hydroxybenzoate hydroxylase for 6- ˚ P212121, with unit-cell parameters a = 62.5, b = 65.9, c = 115.2 A.The azido FAD (Gatti et al., 1994).