Expression, purification and crystallization of an indole prenyltransferase from Aspergillus fumigatus

Residues 38–440 of CdpNPT from A. fumigatus were overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to a resolution of 2.5 Å.

Abstract

CdpNPT from Aspergillus fumigatus is a dimethylallyltryptophan synthase/indole prenyltransferase that catalyzes reverse prenylation at position N1 of tryptophan-containing cyclic dipeptides. Residues 38–440 of CdpNPT were expressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion and microseeding techniques. The crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 84.4, b = 157.1, c = 161.8 Å, α = β = γ = 90.0°.

1. Introduction  

The fungal indole prenyltransfereases (PTs; EC 2.5.1.1; dimethyl­allyltransferases) catalyze the prenylation of tryptophan and its derivatives with dimethylallyl diphosphate (DMAPP) as the prenyl donor in the biosynthesis of structurally diverse prenylated indole alkaloids including ergot alkaloids and terrequinone alkaloids (Steffan et al., 2009 ▶; Li, 2009a ▶,b ▶). The enzymes show varying substrate specificities and regiospecificities for the prenylation of the indole core and the prenyltransfer reactions are classified into two types: ‘regular’ prenylation (attack at the primary centre of the dimethyl­allyl carbocation) and ‘reverse’ prenylation (attack at the tertiary centre). For example, FgaPT2 and FtmPT1 from Aspergillus fumigatus catalyze regular prenylation of l-tryptophan at position C4 and the indole nucleus of the cyclic dipeptide brevianamide F (cyclo-l-­Trp-l-Pro) at position C2 (Fig. 1 ▶ a) to produce the biosynthetic precursors of fumigaclavine C and fumitremorgin B, respectively (Steffan et al., 2007 ▶; Unsöld & Li, 2005 ▶; Grundmann et al., 2008 ▶). In contrast, CdpNPT from A. fumigatus Af293 catalyzes reverse prenylation at position N1 of l-tryptophan and tryptophan-containing cyclic dipeptides such as cyclo-l-Trp-l-Trp and cyclo-l-­Trp-l-Tyr using DMAPP as the prenyl donor (Fig. 1 ▶ b), although the physiological substrate and product of the enzyme reaction have yet to be elucidated (Zou et al., 2009 ▶; Li, 2009a ▶).

Figure 1.

Prenyltransfer reactions catalyzed by FtmPT1 and CdpNPT. (a) Regular prenylation of brevianamide F at position C2 by A. fumigatus FtmPT1. (b) Reverse prenylation of the cyclic dipeptide at position N1 by A. fumigatus CdpNPT.

Although several indole PTs have previously been reported to exist as monomeric proteins (Cress et al., 1981 ▶; Grundmann & Li, 2005 ▶; Steffan et al., 2009 ▶), recent studies on the indole PT enzymes revealed that most of the enzymes form biologically active homodimers composed of approximately 52 kDa subunits in solution (Metzger et al., 2009 ▶; Jost et al., 2010 ▶; Liu & Walsh, 2009 ▶; Steffan et al., 2009 ▶). Furthermore, the X-ray crystal structures of A. fumigatus FgaPT2 and FtmPT1 revealed that the enzymes share the rare (ααββ)₅-barrel fold (Metzger et al., 2009 ▶; Jost et al., 2010 ▶), which was first observed in a bacterial aromatic PT enzyme, NphB, from Streptomyces sp. strain CL190 (Kuzuyama et al., 2005 ▶). The structural analyses also suggested that modification of the active-site cavity results in alteration of the substrate specificities as well as the regiospecificities of the prenylation reactions. Notably, recent site-directed mutagenesis studies of FgaPT2 suggested that the enzyme initially catalyzes reverse prenylation and that a subsequent Cope rearrangement and re-aromatization reactions lead to the regular prenylation of l-tryptophan (Luk et al., 2011 ▶).

CdpNPT (GenBank accession No. AFUA_8G00620), which catalyzes reverse prenylation at position N1 of tryptophan-containing cyclic dipeptides, was identified on chromosome 8 in A. fumigatus Af293 (Zou et al., 2009 ▶; Li, 2009a ▶). The enzyme is composed of 440 amino acids and shares 31 and 28% amino-acid sequence identity with A. fumigatus FgaPT2 and FtmPT1, respectively. A sequence comparison revealed that the dimethylallyl diphosphate-binding residues (Arg100, Tyr187, Tyr261, Lys259, Tyr345, Tyr409 and Tyr413) and the catalytic residue (Glu89) of FgaPT2 and FtmPT1 are well conserved in CdpNPT (the numbering is that of FgaPT2). In contrast, several active-site residues are uniquely altered in CdpNPT. For example, the catalytic Lys174 residue in the FgaPTs, which plays an important role in the Cope rearrangement and re-aromatization reactions (Luk et al., 2011 ▶), is uniquely replaced by Val in CdpNPT. Furthermore, Thr102 and Arg244 in FgaPT2, corresponding to Gly102 and His244, respectively, in FtmPT1, which are thought to determine the substrate specificities of the enzyme reactions (Jost et al., 2010 ▶), are uniquely replaced by Ala residues in CdpNPT. To further clarify the structure–function relationship of the indole PT enzymes and to understand the intimate structural details of the enzyme-catalyzed processes, we expressed a truncated (38–440) His₆-fused A. fumigatus CdpNPT in Escherichia coli and obtained a good-quality crystal of the recombinant enzyme.

2. Experimental  

2.1. Construction of the expression plasmid  

The gene encoding full-length CdpNPT was amplified from the cDNA library of A. fumigatus Af293 (Stratagene) using the forward primer 5′-ATTTATCATATGGACGGTGAGATGACCGCTTCT-3′ and the reverse primer 5′-TTATATCTCGAGTTCAGGCCAAAAGTAATATATAGA-3′ (the NdeI and XhoI restriction sites are indicated in bold). The amplified 1323 bp DNA fragment was digested with NdeI and XhoI and was ligated into the NdeI and XhoI sites of pET22b(+) (Novagen) using a DNA-ligation kit (TaKaRa) for expression as a fusion protein with a His₆ tag at the C-terminus.

The pET22b(+) vector encoding full-length CdpNPT was used as the template to amplify the gene encoding residues 38–440 of CdpNPT by PCR using the forward primer 5′-GGAATTCCATAT GATAGCTTATCATACCCTCAC-3′ and the reverse primer 5′-TTATATCTCGAGTTCAGGCCAAAAGTAATATA-3′ (the NdeI and XhoI restriction sites are indicated in bold). The amplified 1212 bp DNA fragment was digested with NdeI and XhoI and was cloned into the NdeI/XhoI sites of the pET22b(+) expression vector (Novagen) to express the CdpNPT (38–440) mutant enzyme with a His₆ tag at the C-terminus.

2.2. Expression and purification  

After confirmation of their sequences, the pET-22b(+) vectors encoding the full-length CdpNPT and the truncated CdpNPT (38–440) enzymes were independently transformed into E. coli BL21 (DE3) pLysS. The cells harbouring each plasmid were cultured to an OD₆₀₀ of 0.6 in LB medium containing 100 µg ml⁻¹ ampicillin at 303 K. Isopropyl β-d-1-thiogalactopyranoside was then added to a final concentration of 1 mM to induce gene expression and the cultures were incubated for a further 16 h at 303 K.

All of the following procedures were performed at 277 K for both the full-length and the truncated CdpNPTs. The E. coli cells were harvested by centrifugation at 5000g and resuspended in 50 mM Tris–­HCl buffer pH 8.0 containing 0.1 M NaCl and 5%(v/v) glycerol (buffer A). The cells were disrupted by sonication six times for 10 s each with 15 s intervals between each sonication and the lysate was centrifuged at 12 000g for 30 min. The supernatant thus obtained was loaded onto an Ni Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with buffer A. After washing the column with 50 mM Tris–HCl buffer pH 7.5 containing 200 mM NaCl and 5%(v/v) glycerol (buffer B), the His₆-tagged CdpNPT was subsequently eluted with buffer B containing 500 mM imidazole. The protein solution was then diluted fivefold with 50 mM Tris–HCl buffer pH 7.5 containing 5%(v/v) glycerol and 2 mM DTT (buffer C) and applied onto a Resource-Q column (GE Healthcare). The column was washed with buffer C containing 100 mM NaCl and the protein was subsequently eluted at 160 mM NaCl using a linear gradient of 100–500 mM NaCl. The protein solution was further purified to homogeneity by gel-filtration chromatography on Superdex 200 HR (10/300 GL; GE Healthcare) and was concentrated to 10 mg ml⁻¹ in 20 mM Tris–HCl pH 7.5 containing 100 mM NaCl and 2 mM DTT. Dynamic light-scattering (DLS) analysis was performed using a DynaPro MSXTC molecular-sizing instrument (Protein Solutions Inc.). After centrifugation using a 0.22 µm Ultrafree-MC filter (Millipore) to remove particulate material from the protein solution, the solution properties of the purified protein were monitored. Data were acquired from 50 scattering measurements at 278 K; a total of three sets of data were analyzed using the DYNAMICS software package (Protein Solutions Inc.) and were averaged.

2.3. Crystallization and X-ray data collection  

Initial crystallization attempts were performed at 278 and 293 K using the sitting-drop vapour-diffusion method by mixing 0.8 µl of either the purified full-length CdpNPT solution or the truncated His₆-tagged CdpNPT (38–440) solution with an equal volume of reservoir solution (using Wizard I and II from Emerald BioSystems and Crystal Screen and Crystal Screen II from Hampton Research) and equilibrating the mixture against 100 µl reservoir solution. Clusters of crystals were observed a week later using a crystallization solution consisting of 100 mM Tris–HCl pH 8.5, 200 mM lithium sulfate, 1.26 M ammonium sulfate. Further crystallization was attempted at various pH values, together with the use of 1.0–2.0 M ammonium sulfate, 200 mM lithium sulfate as precipitant, but diffraction-quality crystals were not obtained. Therefore, further optimization of the crystallization conditions was performed using the microseeding technique. The crystal clusters in the drop were crushed with a needle to produce a stock mixture of crystal seeds, which was then diluted 10³-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold and 10⁷-­fold with reservoir solutions at various pH values, together with the use of 1.0–2.0 M ammonium sulfate, 200 mM lithium sulfate, 2 mM l-tryptophan as precipitant. An aliquot of 0.8 µl of each seed solution was subsequently mixed with an equal volume of the purified enzyme solution and was again screened against the reservoir solutions. Diffraction-quality crystals were finally obtained using the His₆-tagged CdpNPT (38–440) enzyme solution at 293 K in 100 mM MES pH 6.5 containing 1.1 M ammonium sulfate, 150 mM lithium sulfate and 2 mM l-trypto­phan using the sitting-drop vapour-diffusion method and the seeding technique.

The crystals were transferred into a cryoprotectant consisting of 100 mM MES pH 6.5, 1.1 M ammonium sulfate, 150 mM lithium sulfate, 2 mM l-tryptophan, 20%(v/v) glycerol. After a few seconds, the crystals were picked up in a nylon loop and flash-cooled at 100 K in a nitrogen-gas stream. X-ray diffraction data sets were collected from a single crystal on beamline X06DA at the Swiss Light Source using a MAR 225 CCD detector with a crystal-to-detector distance of 210 mm. The wavelength of the synchrotron radiation was 1.0000 Å. A total of 200 frames were recorded with a 0.5° oscillation angle and a 1 s exposure time. The data were indexed, integrated and scaled using the HKL-2000 program package (Otwinowski & Minor, 1997 ▶).

3. Results and discussion  

Our initial crystallization trials using the full-length His₆-tagged CdpNPT did not yield any diffraction-quality crystals. This may be attributed to the high flexibility of the 37 N-terminal amino-acid residues, as predicted by a DNASTAR protein-chain flexibility search using the Karplus–Schulz method (flexibility threshold value = 1.0; Burland, 2000 ▶). Therefore, it was anticipated that truncation of the N-­terminal amino-acid residues of CdpNPT would facilitate the generation of better diffracting crystals. The truncated His₆-tagged CdpNPT (38–440) was thus constructed and expressed in E. coli. The truncated enzyme exhibited identical prenylation activity for l-­tryptophan to that of the full-length enzyme (data not shown). The typical yield was about 10 mg per litre of culture. The truncated His₆-tagged CdpNPT (38–440) migrated as a single band with a molecular mass of 47 kDa on SDS–PAGE, which agrees well with the calculated value of 46.7 kDa. In contrast, the recombinant enzyme yielded a molecular mass of 96 kDa in a gel-filtration experiment. Furthermore, DLS analysis after gel filtration revealed a monomodal distribution with a polydispersity value of 22.8% and an estimated molecular mass of 82 kDa, suggesting that the truncated His₆-tagged CdpNPT (38–440) also forms a homodimer, as in the case of the full-length CdpNPT. In contrast, truncation of the N-terminal 43 residues of CpaD, another fungal indole PT from Aspergillus sp., reportedly resulted in disruption of both the dimerization and catalytic activity (Liu & Walsh, 2009 ▶).

Crystals appeared reproducibly within a week and the largest crystal grew to dimensions of approximately 0.45 × 0.25 × 0.08 mm (Fig. 2 ▶). A complete data set was collected to 2.5 Å resolution. Detailed data-processing statistics are shown in Table 1 ▶. Based on the diffraction data, the space group was determined to be P2₁2₁2₁, with unit-cell parameters a = 84.4, b = 157.1, c = 161.8 Å, α = β = γ = 90.0°. With four monomers in the asymmetric unit, the Matthews volume (V _M; Matthews, 1968 ▶) was calculated to be 2.9 ų Da⁻¹ and the estimated solvent content was 56.5%, which is in the range normally observed for protein crystals. Structure determination using the molecular-replacement method with the MOLREP program (Vagin & Teplyakov, 2010 ▶) and a CdpNPT (38–440) structure model generated by the SWISS-MODEL package (http://swissmodel.expasy.org) based on the crystal structure of A. fumigatus FgaPT2 (PDB entry 3i4x; Metzger et al., 2009 ▶) as a search model is now in progress. Simultaneously, we are also attempting to crystallize the truncated His₆-tagged CdpNPT (38–440) complexed with substrate analogues. These structural analyses will provide valuable insights into the catalytic mechanism of the indole PT enzymes.

Figure 2.

Crystals of the truncated His₆-tagged recombinant CdpNPT (38–440). The dimensions of the crystals are approximately 0.45 × 0.25 × 0.08 mm.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

Beamline	X06DA, Swiss Light Source
Wavelength (Å)	1.00000
Temperature (K)	100
Detector	MAR 225 CCD
Crystal-to-detector distance (mm)	210
Oscillation angle (°)	0.5
No. of frames	200
Exposure time (s)	1
No. of crystals	1
Space group	P212121
Unit-cell parameters
a (Å)	84.4
b (Å)	157.1
c (Å)	161.8
α = β = γ (°)	90
Resolution (Å)	50.0–2.5 (2.59–2.50)
Total reflections	309778
Unique reflections	75494
Mosaicity (°)	0.27
Multiplicity	4.1 (3.8)
Completeness (%)	99.6 (96.8)
〈I/σ(I)〉	23.8 (3.3)
Rmerge† (%)	6.0 (31.9)

^† R _merge = , where I(hkl) is the intensity of reflection hkl, is the sum over all reflections and is the sum over i measurements of reflection hkl.