Structural Basis of Tryptophan Reverse N-Prenylation Catalyzed by

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Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Structural Basis of Tryptophan Reverse N‑Prenylation Catalyzed by CymD Benjamin W. Roose and David W. Christianson* Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States

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S Supporting Information *

ABSTRACT: Indole prenyltransferases catalyze the prenylation of Ltryptophan (L-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants, many of which possess useful biological properties. Among this family of enzymes, CymD from Salinispora arenicola catalyzes the reverse N1 prenylation of L-Trp, an unusual reaction given the poor nucleophilicity of the indole nitrogen. CymD utilizes dimethylallyl diphosphate (DMAPP) as the prenyl donor, catalyzing the dissociation of the diphosphate leaving group followed by nucleophilic attack of the indole nitrogen at the tertiary carbon of the dimethylallyl cation. To better understand the structural basis of selective indole N-alkylation reactions in biology, we have determined the X-ray crystal structures of CymD, the CymD−L-Trp complex, and the CymD−L-Trp−DMSPP complex (DMSPP is dimethylallyl S-thiolodiphosphate, an unreactive analogue of DMAPP). The orientation of L-Trp with respect to DMSPP reveals how the active site contour of CymD serves as a template to direct the reverse prenylation of the indole nitrogen. Comparison to PriB, a C6 bacterial indole prenyltransferase, offers further insight regarding the structural basis of regioselective indole prenylation. Isothermal titration calorimetry measurements indicate a synergistic relationship between L-Trp and DMSPP binding. Finally, activity assays demonstrate the selectivity of CymD for L-Trp and indole as prenyl acceptors. Collectively, these data establish a foundation for understanding and engineering the regioselectivity of indole prenylation by members of the prenyltransferase protein family.

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the structural and enzymatic properties of members of the DMATS group has been an active area of investigation given their role in the biosynthesis of useful natural products.15−17 There is currently a need for additional structural studies to better inform molecular modeling of this class of enzymes. Nature frequently employs tryptophan as a template for the biosynthesis of natural products owing to the reactivity of its indole ring.18−20 Indoles readily undergo electrophilic aromatic substitution reactions, including alkylation (e.g., prenylation),21 nitration,22 and halogenation,23 to yield a variety of bioactive natural products, including antibiotics, anticancer agents, herbicides, anti-inflammatories, and antifungal compounds.24 The past decade has seen the discovery of indole prenyltransferases capable of prenylating L-Trp and indole derivatives at all seven sites of the indole moiety (Figure 1).8,25 In 2010, Schultz and co-workers reported the characterization of CymD from the marine actinobacterium Salinispora arenicola, the first bacterial tryptophan prenyltransferase to be identified.26 CymD adopts the canonical ABBA fold based on its primary structure, though it shares only ∼25% sequence identity with other subsequently discovered ABBA fold

renylated natural products often exhibit useful cytotoxic, antimicrobial, and antioxidant properties that are not observed in their nonprenylated precursors.1,2 In plants, fungi, and bacteria, prenylation is catalyzed by enzymes known as prenyltransferases.3 These enzymes catalyze the transfer of a prenyl moiety from donors such as dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), or farnesyl diphosphate (FPP) to prenyl acceptors ranging from small molecule metabolites4 and peptides5 to proteins6 and nucleic acids.7 Prenylation can occur via the primary carbon of the allylic diphosphate donor (normal prenylation) or via the tertiary carbon (reverse prenylation).8 Aromatic prenyltransferases exist in both membrane-bound9 and soluble forms.10 Soluble aromatic prenyltransferases share a common αββα or ABBA fold, also known as a PT barrel, consisting of a central core of 10 antiparallel β-strands surrounded by 10 α-helices.11−14 Though their tertiary structure is conserved, the soluble aromatic prenyltransferases can be further divided into two groups on the basis of their primary structure: (1) the NphB/CloQ group, members of which prenylate naphthalenes, quinones, phenols, and phenazines, and (2) the dimethylallyltryptophan synthase (DMATS) group, members of which prenylate indole derivatives, tyrosine, naphthalenes, and xanthones.3 Members of the DMATS group are found in both bacteria and fungi, though their level of sequence identity is low. Characterizing © XXXX American Chemical Society

Received: May 2, 2019 Revised: June 15, 2019 Published: June 28, 2019 A

DOI: 10.1021/acs.biochem.9b00399 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Prenylation sites of L-tryptophan, with representative prenyltransferases from bacteria (blue) and fungi (red) noted for each site: CymD from Salinispora arenicola,26 FtmPT2 from Aspergillus f umigatus,27 FtmPT1 from A. f umigatus,28 KgpF from Microcystis aeruginosa,29 AnaPt from Nassarius f ischeri,30 FgaPT2 from A. f umigatus,31 SCO7467 from Streptomyces coelicolor,32 5-DMATS from Aspergillus clavatus,25 PriB from Streptomyces sp. RM-5-8,33 TleC from Streptomyces blastmyceticus,34 and 7-DMATS from A. f umigatus.35 Shown is the reverse N-prenylation reaction catalyzed by CymD to produce N-DMAT.

Gene Cloning. The codon-optimized cymD gene from S. arenicola CNS-205 (GenBank accession no. ABW00334.1; UniProt accession no. A8M6W6) was purchased from GenScript. The full-length cymD gene (minus the start methionine) was amplified by polymerase chain reaction using the forward primer 5′-TACTTCCAATCCAATGCAACCGAGGAGCTGACCACC-3′ and the reverse primer 5′-TTATCCACTTCCAATGTTATTACTCGGTGCGACCACGCGC-3′. The amplified cymD gene was inserted using ligation-independent cloning (LIC) into a pET-His6-GFPTEV-LIC vector, a gift from S. Gradia acquired from Addgene (plasmid 29663). The resulting pET-His6-GFP-TEV-CymD plasmid was amplified in NEB-5α competent Escherichia coli cells (New England Biolabs), purified using a Monarch plasmid miniprep kit (New England Biolabs), and sequenced at the University of Pennsylvania DNA Sequencing Facility to verify the integrity of the cymD gene. Protein Preparation. The pET-His6-GFP-TEV-CymD plasmid was transformed into BL21(DE3) competent E. coli cells (New England Biolabs) and cultured overnight at 37 °C on an LB-agar culture plate supplemented with 50 μg/mL kanamycin. Single colonies of transformed cells were used to inoculate 5 mL cultures of Terrific Broth (TB) medium supplemented with 50 μg/mL kanamycin that were then incubated overnight at 37 °C while being shaken at 250 rpm. The 5 mL cultures were used to inoculate 6 × 1 L of TB medium supplemented with 50 μg/mL kanamycin. The 1 L cultures were incubated at 37 °C while being shaken at 250 rpm until the OD600 reached ∼1. Protein expression was induced by adding isopropyl β-L-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The induced cultures were incubated overnight at 25 °C while being shaken at 250 rpm. Cells were pelleted by centrifugation and stored at −80 °C prior to purification. Cell pellets were thawed and resuspended in 20 mM sodium phosphate (pH 7.4), 500 mM NaCl, 1 mM tris(2carboxyethyl)phosphine hydrochloride (TCEP-HCl), and 20 mM imidazole. The cell suspension was treated with 1 mM EDTA, 1 mg/mL lysozyme, 1 kilounit of benzonase, and protease inhibitor (Roche) and incubated for 1 h at 4 °C. Cells were then lysed by sonication followed by the addition of 2 mM MgCl2. The cell lysate was clarified by centrifugation at 38000g for 30 min at 4 °C. The supernatant was loaded onto a 5 mL HisTrap HP column (GE Healthcare) and eluted via a 20 column volume gradient to 20 mM sodium phosphate (pH

bacterial prenyltransferases. CymD catalyzes the reverse prenylation of the indole nitrogen of L-tryptophan and does so in a cation-independent fashion. CymD is encoded by the cymD gene of the cym gene cluster that is responsible for the biosynthesis of the cyclic peptides cyclomarin A and cyclomarazine A, potent anti-inflammatory and antimicrobial compounds, respectively.36 CymD exclusively utilizes DMAPP as the prenyl donor, as the use of isoprenyl, geranyl, or farnesyl disphosphate yields no detectable products.26 Qian and coworkers report that the mechanism of CymD proceeds through a dissociative mechanism in which the dimethylallyl cation is formed in the first step of catalysis.37 In the second step, deprotonation of the indole nitrogen by a general base is proposed to precede or coincide with nucleophilic attack by the indole nitrogen at the tertiary carbon of the dimethylallyl cation to yield N-dimethylallyl-L-tryptophan (N-DMAT). Here, we report the 1.33 Å resolution crystal structure of CymD complexed with L-Trp and dimethylallyl S-thiolodiphosphate (DMSPP), an unreactive analogue of DMAPP. This ternary complex reveals the orientation of the prenyl donor and acceptor during catalysis and supports the proposed mechanism by which prenylation proceeds via direct nucleophilic attack by the indole nitrogen, rather than via a normal C3 prenylation followed by an aza-Cope rearrangement, an unlikely but plausible alternative reaction pathway.37−39 Additional structures of CymD include the native (i.e., substrate-free) enzyme at 1.70 Å resolution and the complex with L-Trp at 1.66 Å resolution. Activity assays and isothermal calorimetry experiments offer additional insight into the substrate scope of CymD as well as the thermodynamics of ligand binding in the active site.

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MATERIALS AND METHODS Reagents. Unless specified, chemicals used for protein expression and purification were purchased from Fisher, Sigma, RPI, or GoldBio and used without further purification. Reagents used for crystallization were purchased from Hampton Research. L-Tryptophan (99%), D-tryptophan (99%), indole (>99%), indoline (98%), benzothiazole (97%), benzoxazole (>99%), benzimidazole (98%), aniline (>99%), Lglutamine (99%), and L-asparagine (99%) were purchased from Acros Organics. Dimethylallyl diphosphate (DMAPP) (>95%) and dimethylallyl S-thiolodiphosphate (DMSPP) (>95%) were purchased from Echelon Biosciences. B


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Biochemistry 7.4), 500 mM NaCl, 1 mM TCEP-HCl, and 500 mM imidazole. The protein was treated with 3 mg of TEV protease to cleave the GFP tag and concurrently dialyzed into 20 mM sodium phosphate (pH 7.4), 500 mM NaCl, 1 mM TCEPHCl, and 20 mM imidazole at 4 °C. The protein digest was loaded onto a 5 mL HisTrap HP column, and flow-through fractions containing pure CymD were collected and pooled. CymD was concentrated to ∼5 mL using an Amicon Ultra-15 filter (molecular weight cutoff of 30 kDa) and loaded onto a HiLoad 26/600 Superdex 200 pg size-exclusion column (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, and 1 mM TCEP-HCl. Elution fractions containing CymD were identified by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) (Figure S1), pooled, and concentrated to ∼10 mg/mL. Crystallization. Crystals of CymD complexed with L-Trp and DMSPP were grown by the sitting drop vapor diffusion method at 21 °C. A 100 nL drop of the protein solution [9.4 mg/mL CymD, 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM TCEP-HCl, 2 mM L-Trp, and 2 mM DMSPP] was added to 100 nL of the precipitant solution [1% (w/v) tryptone, 0.05 M sodium 4-(2-hydroxyethyl)piperazine-1-ethanesulfonate (HEPES-Na) (pH 7.0), and 12% PEG 3350] and equilibrated against a 50 μL reservoir of the precipitant solution. Crystals of native CymD were grown by the sitting drop vapor diffusion method at 4 °C. A 2 μL drop of the protein solution [9.3 mg/ mL CymD, 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, and 1 mM TCEP-HCl] was added to a 2 μL drop of the precipitant solution [4% 2-propanol, 0.1 M Bis-Tris propane (pH 9.0), and 20% PEG monomethyl ether 5000] and equilibrated against a 500 μL reservoir of the precipitant solution. The sitting drop was streak seeded with microcrystals of CymD grown in the same precipitant. Crystals of CymD complexed with L-Trp were grown by the sitting drop vapor diffusion method at 21 °C. A 2.5 μL drop of the protein solution [9.8 mg/mL CymD, 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM TCEP-HCl, and 2 mM L-Trp] was added to a 2.5 μL drop of the precipitant solution [0.04 M citric acid, 0.06 M Bis-Tris propane (pH 6.4), and 20% PEG 3350] and equilibrated against a 400 μL reservoir of the precipitant solution. All CymD crystals were briefly immersed in the cryoprotectant solution (mother liquor supplemented with either 25% ethylene glycol or 25% glycerol) before being flashcooled in liquid nitrogen. Data Collection and Structure Determination. X-ray diffraction data from crystals of the CymD−L-Trp−DMSPP complex were collected on beamline 17-ID-2 (FMX) at National Synchrotron Light Source II (NSLS-II) at Brookhaven National Laboratory. Diffraction data from CymD crystals were collected remotely on Northeastern Collaborative Access Team (NE-CAT) beamline 24-ID-C at the Advanced Photon Source (APS). Diffraction data from crystals of the CymD−L-Trp complex were collected remotely on beamline 12-2 at the Stanford Synchrotron Radiation Laboratory (SSRL) at Stanford University. Diffraction data were indexed and integrated using either iMosflm40 or XDS41 and scaled using Aimless42 in the CCP4 program suite.43 Although some diffraction data sets were characterized by high Rmerge values, the precision−indicating−merging R factor (Rpim), which reflects the increased accuracy of highly redundant data sets, was satisfactory for all shells of data in all data sets. Diffraction data collection statistics are listed in Table 1.

Table 1. Data Collection and Refinement Statistics CymD space group a, b, c (Å) α, β, γ (deg) beamline wavelength (Å) resolution (Å) total no. of reflections no. of unique reflections Rmergea,b Rpima,c CC1/2a,d I/σ(I)a redundancya completeness (%)a no. of reflections used in refinement/test set Rworke Rfreef no. of protein chains no. of non-hydrogen atoms protein ligand ion solvent average B factor (Å2) protein ligand ion solvent root-mean-square deviation from ideal geometry bonds (Å) angles (deg) Ramachandran plot (%)g favored allowed outliers MolProbity score PDB entry

CymD−L-Trp complex

Unit Cell I4 P212121 157.7, 157.7, 85.3, 86.3, 56.3 141.7 90, 90, 90 90, 90, 90 Data Collection APS 24-ID-C SSRL 12-2 0.91950 55.8−1.70 1209693 76243

0.97946 86.3−1.66 681939 123383

CymD−L-Trp− DMSPP complex I4 129.4, 129.4, 49.8 90, 90, 90 NSLS-II 17-ID-2 0.97932 29.1−1.33 708407 95021

0.226 (1.072) 0.104 (0.360) 0.083 (0.394) 0.070 (0.251) 0.994 (0.742) 0.986 (0.886) 8.0 (2.9) 8.9 (3.5) 15.9 (16.0) 5.5 (5.6) 99.9 (100) 99.6 (99.7) Refinement 76236/1996 123289/2000

0.064 (0.623) 0.037 (0.375) 0.999 (0.806) 15.0 (2.3) 7.5 (7.0) 99.8 (97.3)

0.167 0.208 2

0.163 0.192 3

0.166 0.181 1

5266 46 − 649

7951 62 − 905

2814 38 3 411

23 30 − 32

18 18 − 30

17 16 15 31

0.007 0.8

0.006 0.8

0.005 0.9

97.5 2.5 0 1.12 6OS3

98.4 1.6 0 1.08 6OS5

97.5 2.5 0 0.97 6OS6

95019/1993

a

Values in parentheses refer to the highest-resolution shell of the data. Rmerge = ∑|Ih − ⟨Ih⟩|/∑⟨Ih⟩, where Ih is the intensity measure for reflection h and ⟨Ih⟩ is the average intensity for reflection h calculated from replicate data. cRpim = ∑[1/(n − 1)1/2]|Ih − ⟨Ih⟩|/∑⟨Ih⟩, where n is the number of observations (redundancy). dCC1/2 = στ2/(στ2 + σε2), where στ2 is the true measurement error variance and σε2 is the independent measurement error variance. eRwork = ∑||Fo| − |Fc||/∑ |Fo| for reflections contained in the working set. |Fo| and |Fc| are the observed and calculated structure factor amplitudes, respectively. fRfree = ∑||Fo| − |Fc||/∑|Fo| for reflections contained in the test set held aside during refinement. gAssessed by MolProbity. b

The crystal structure of the CymD−L-Trp−DMSPP complex was determined by molecular replacement using C


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Figure 2. (a) Crystal structure of CymD complexed with L-Trp and DMSPP illustrating the ABBA fold with a central active site cavity. (b) Stereoview of the active site of CymD, with selected side chains (sticks) and water molecules (red spheres) shown. Hydrogen bonds are represented by black dashed lines. The interaction between the indole nitrogen and general base E64 is colored cyan. Polder omit maps corresponding to L-Trp and DMSPP are shown as green mesh and contoured at 5σ. (c) Offset π-stacking interactions in the active site of CymD. Hydrogen bonds are shown as dashed black lines. Interplanar distances between π systems (green dashes) are indicated (angstroms). The angle between the indole nitrogen and dimethylallyl plane is colored pink.

Phaser.44 A search model based on the crystal structure of unliganded PriB prenyltransferase [Protein Data Bank (PDB) entry 5JXM]33 was generated using the SWISS-MODEL server45 and converted to a poly-ALA model using Chainsaw46 in the CCP4 program suite. Iterative cycles of refinement and manual model building were performed using Phenix47 and WinCoot,48 respectively. Translation−libration−screw (TLS) refinement was performed during the later stages of refinement using TLS groups determined by Phenix. Disordered protein side chain atoms showing no electron density in the 2Fo − Fc map were deleted from the model, and electron density peaks that were not confidently interpretable were left unmodeled. Refinement proceeded until Rfree converged at its lower limit. The quality of the final model was assessed using MolProbity.49 Refinement statistics are listed in Table 1. All structural figures were generated using PyMOL.50 Root-meansquare deviation (rmsd) values were calculated using Superpose51,52 in the CCP4 program suite. The refined protein structure of the CymD−L-Trp−DMSPP complex, without the ligands and solvent, was used as the search model for phasing the initial electron density maps of native CymD and the CymD−L-Trp complex. These structures were refined and evaluated following the same procedure outlined above for the CymD−L-Trp−DMSPP complex. All refinement statistics are listed in Table 1. Enzyme Kinetics. The prenyltransferase activity of CymD was measured using the EnzChek Pyrophosphate Assay Kit

(Invitrogen). The reaction buffer was composed of 50 mM Tris-HCl (pH 7.5), 1 mM MgCl2, and 0.1 mM sodium azide. The 100 μL reaction mixture contained (final concentrations) 200 μM 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), 0.1 unit of purine nucleoside phosphorylase (PNPase), 2 units of inorganic pyrophosphatase (IPPase), 1 μM CymD, and 80 μM DMAPP. The assay was initiated by adding the prenyl acceptor substrate to a final concentration of 500 μM. The reaction was monitored in triplicate in a flatbottom transparent 96-well plate using a Tecan M1000 spectrophotometer. The reaction velocity was calculated from the increase in A360 within the linear range. Isothermal Titration Calorimetry. The thermodynamics of binding of L-Trp and DMSPP to CymD were measured using a MicroCal iTC 200 isothermal titration calorimeter (GE Healthcare); 300 μM L-Trp or DMSPP was titrated into 30 μM CymD in 20 mM Tris-HCl, 200 mM NaCl, 1 mM TCEPHCl, and 10% (v/v) glycerol at 25 °C. Enthalpogram data were visualized and analyzed using Origin (OriginLab, Northampton, MA).

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RESULTS AND DISCUSSION X-ray Crystallography. Crystal structures of CymD were determined and refined to Rwork and Rfree values ranging from 0.163 to 0.167 and from 0.181 to 0.208, respectively. The low MolProbity scores indicate no significant errors in the protein models. The structures of native CymD and the CymD−L-Trp D


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Biochemistry complex were quite similar to the structure of the CymD−LTrp−DMSPP complex, with rmsd values of 0.79 Å for 344 Cα atoms and 0.67 Å for 354 Cα atoms, respectively. CymD is a monomer in three different crystal forms on the basis of analysis with PISA,53 consistent with size-exclusion chromatography of the enzyme in solution. The ternary complex of CymD bound to L-Trp and DMSPP crystallizes in space group I4 with one protein chain per asymmetric unit. Single molecules of L-Trp and DMSPP are bound in the enzyme active site, located in the central β-barrel of the ABBA fold (Figure 2a). The carboxylate Oε1 atom of E64, which acts as a general base in catalysis, is positioned 2.8 Å from the indole nitrogen of L-Trp with a syn orientation (Figure 2b). CymD makes only one other direct interaction with L-Trp, a 2.6 Å hydrogen bond donated by Y326 to the LTrp carboxylate. However, there are several additional watermediated hydrogen bonds between CymD and L-Trp. The indole ring of L-Trp forms an offset π-stacking interaction with the side chain of Y326 (Figure 2c). The side chains of Y274 and Y341 are oriented in perpendicular fashion to the indole ring of L-Trp, and F192 and Y209 are nearby. These residues form an aromatic box to accommodate and orient the indole ring of L-Trp. The crystal structure of the CymD−L-Trp−DMSPP complex mimics that of the precatalytic Michaelis complex, revealing the spatial relationship between L-Trp and the dimethylallyl moiety prior to catalysis. The DMSPP molecule lies parallel to the L-Trp indole ring, with the dimethylallyl moiety positioned 3.7 Å away. The tertiary dimethylallyl carbon is optimally positioned for nucleophilic attack by the indole nitrogen. The nitrogen is positioned 3.4 Å away and oriented at an angle of 104° relative to the dimethylallyl plane, close to the Bürgi−Dunitz angle of 107°.54 Notably, the primary dimethylallyl carbon is 5.9 Å from the indole C3 atom, effectively ruling out the possibility of an aza-Cope rearrangement mechanism and affirming the direct N-prenylation mechanism proposed by Qian and colleagues. 37 The dimethylallyl moiety of DMSPP is parallel to the indole ring of W148, which likely stabilizes the dimethylallyl cation intermediate via a cation−π interaction during catalysis. The thiolodiphosphate group of DMSPP lies within a positively charged pocket in the CymD active site, with the β-phosphate forming salt bridges with the side chains of R205, K207, R337, and K339. The α-phosphate, on the other hand, forms mostly hydrogen bonds with CymD, receiving hydrogen bonds from the side chains of Q77, Y274, and Y341, and forms a single salt bridge with K146 (Figure 2b). The crystal structures of native CymD and the CymD−LTrp complex were solved to determine the extent of substrateinduced conformational changes in the CymD active site (Figure 3). Native CymD crystallizes with two monomers in the asymmetric unit. Both CymD monomers show a molecule of bis-tris propane bound in the active site, with an amino NH group donating a 2.8 Å hydrogen bond to E64 in chain B (this distance is 3.3 Å in chain A). Structural comparison of native CymD and the CymD−L-Trp−DMSPP complex shows that the active site side chains near L-Trp do not substantially reorient to bind the substrate. Even in the absence of L-Trp, the E64 carboxylate is perfectly preoriented and poised to deprotonate the indole nitrogen, and the Y326 side chain is positioned for offset π−π stacking. Inspection of the diphosphate-binding channel, however, reveals that several

Figure 3. (a) Stereoview of the active site pocket in native CymD (gray) and CymD complexed with L-Trp and DMSPP (purple). (b) Stereoview of the active site pocket in CymD complexed with L-Trp and benzoic acid (cyan) and CymD complexed with L-Trp and DMSPP (purple).

positively charged side chains reorient to form salt bridges upon DMSPP binding. The CymD−L-Trp complex crystallizes with three monomers in the asymmetric unit. The active site of chain A contains bound L-Trp as well as what appears to be a molecule of benzoic acid stacked roughly 4.0 Å above the indole ring of L-Trp (Figure 3). Benzoic acid was not present in either the protein or precipitant solutions, so its origin is unclear. Benzoic acid accepts hydrogen bonds from Y274 (2.5 Å), Y209 (2.7 Å), and Q77 (3.0 Å) and also makes an offset π−π stacking interaction (4.1 Å) with W148. Similarly, the active site in chain B contains bound L-Trp along with what appears to be a molecule of imidazole. Like benzoic acid, the imidazole forms π−π interactions with the indole group of L-Trp (3.8 Å) and the side chain of W148 (4.1 Å). Additionally, imidazole forms a 2.7 Å hydrogen bond with a bound diphosphate molecule. Chain C shows positive electron density in the active site, though it does not fit any small molecules present in the crystallization drop (e.g., bis-tris propane and citrate) and was thus deemed uninterpretable and left unmodeled. The binding of benzoic acid and imidazole in chains A and B indicates that the offset π−π stacking network in the CymD active site is capable of binding other aromatic small molecules besides the dimethylallyl carbocation and indicates the potential of expanding the chemistry of electrophilic aromatic substitution reactions catalyzed by CymD. CymD Sequence Analysis and Structural Relationships. The amino acid sequence of CymD was aligned58 with sequences of several other bacterial indole prenyltransferases sharing the ABBA fold to identify key conserved residues as well as highlight differences that could explain the regioselectivity of each prenyltransferase (Figure S2 and Table S1). The sequence of CymD is 45% identical with that of IlaO from Streptomyces atratus, another reverse L-Trp E


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Figure 4. Comparison of the orientation of L-Trp and DMSPP between CymD and PriB. (a) Stereoview of L-Trp and DMSPP bound to CymD (purple) and PriB (green). (b) DMSPP orientation viewed along the z-axis. (c) Connolly surfaces of DMSPP and nearby side chains showing the repositioning of the dimethylallyl moiety resulting from alternative reaction templates.

N-prenyltransferase located in the ila gene cluster responsible for the biosynthesis of ilamycin.55 The sequence of CymD, however, is only 25−26% identical with those of C5 prenyltransferase SCO7467,32 C6 prenyltransferases PriB33 and IptA,56 and C7 prenyltransferases MpnD57 and TleC.34 There are several conserved residues among the bacterial indole prenyltransferases. Notably, the general base E64 is conserved, highlighting its key role in indole activation.33,59 The aromaticity of residue 326 is conserved (Tyr in the N, C5, and C6 prenyltransferases; Phe in the C6 prenyltransferases) as well as the aromaticity of residues 209 (Tyr), 274 (Tyr or His), and 341 (Tyr) that comprise the aromatic box in the active site. Several residues lining the diphosphate-binding pocket of the active site are conserved as well, notably K146, R205, R337, and Y341. These residues make direct contacts with the substrate diphosphate group in the CymD−L-Trp−DMSPP complex. This observation supports the hypothesis that indole bacterial prenyltransferases have evolved to fix the position of the prenyl diphosphate group, with prenyl acceptor specificity and prenylation regioselectivity dictated by subtle variations in the residues that interact with and align the dimethylallyl moiety and prenyl acceptor in the active site.59−61 Structural comparison of the ternary CymD−L-Trp− DMSPP complex and the 1.4 Å resolution structure of the C6 PriB−L-Trp−DMSPP complex (PDB entry 5INJ)33 offers considerable insight regarding the relationship between active site architecture and prenylation regioselectivity. PriB catalyzes the normal prenylation of L-Trp at C6, and while its sequence

is only 25% identical with that of CymD, both enzymes share the same ABBA fold (the rmsd of 319 Cα atoms is 1.8 Å). The orientation of L-Trp is largely conserved between both enzymes (Figure 4a), though PriB forms four direct contacts with L-Trp, whereas CymD forms only two. In both enzymes, there is a tyrosine side chain parallel to L-Trp that stabilizes the prenyl acceptor via offset π−π stacking as well as a hydrogen bond with the α-carboxylate group of L-Trp. The dimethylallyl moieties of bound DMSPP, however, are positioned differently in CymD and PriB relative to L-Trp (Figure 4b). In CymD, the tertiary dimethylallyl carbon is 3.4 Å from N1, whereas in PriB, the primary dimethylallyl carbon is 3.6 Å from C6. This shift in dimethylallyl position results from differences in the side chains lining the dimethylallyl pocket (Figure 4c). A79 in CymD is substituted with L110 in PriB, with the longer side chain effectively pushing the dimethylallyl group closer to the C6 atom of L-Trp. Likewise, on the opposite side, the L193 side chain pushes the dimethylallyl moiety in CymD toward the N1 position of LTrp, while the perpendicular F220 in PriB allows the dimethylallyl to shift closer to the C6 position. M132 in CymD and W165 in PriB also make favorable contacts with the two methyl carbons of DMSPP, further fixing its position over L-Trp. Finally, the aromatic capping group stabilizing the carbocation during catalysis is different between the enzymes. The larger tryptophan side chain in CymD (W148) allows the dimethylallyl to move closer to the L-Trp nitrogen, whereas in PriB, the smaller tyrosine keeps it closer to C6 of L-Trp. F


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Biochemistry

sulfoxide (DMSO)]63 resulting from the reduction of aromaticity adjacent to the nitrogen. The compounds benzimidazole, benzoxazole, and benzothiazoleindole analogues with C3 substituted with nitrogen, oxygen, and sulfur, respectivelyshowed very little prenylation activity. The very low reactivity of benzothiazole can be attributed to decreased aromaticity caused by the poor overlap of the sulfur 3p orbitals and 2p orbitals of the adjacent carbons. The very low level of benzimidazole prenylation, however, is perplexing. Its nitrogen (pKa = 16.4 in DMSO) is more acidic than that of indole (pKa = 21.0 in DMSO),64 and solvent isotope studies performed by Qian and co-workers identified nitrogen deprotonation as the rate-limiting step in catalysis.37 Thus, from a thermodynamic perspective, benzimidazole would be expected to show an increased rate of prenylation by DMAPP. On the other hand, the nucleophilicity of the deprotonated benzimidazole nitrogen is likely decreased by the presence of the additional nitrogen atom in the aromatic π system. Similarly, the electronegative oxygen in benzoxazole likely decreases the nucleophilicity of the deprotonated N1 atom. These data highlight the unique versatility of indole as a biosynthetic precursor; its nitrogen can act as an acid while still retaining its nucleophilic character. The L-amino acids glutamine and asparagine were also tested for prenylation activity because the pKa of their carboxamide nitrogens is ∼25 (in DMSO),65 roughly comparable to the indole nitrogen. Furthermore, in the case of glutamine, the side chain NH2 group is approximately isosteric with the indole NH group of tryptophan. Neither L-Gln nor L-Asn showed any prenylation activity. Bis-tris propane was also evaluated because it occupies the active site of the native CymD crystal structure with one of its amino groups donating a hydrogen bond to E64. Bis-tris propane showed very little activity, indicating that while flexible nitrogen-containing small molecules may potentially occupy the active site, they do not act as suitable prenyl acceptors.

Collectively, these subtle differences in active site architecture direct regioselectivity by sterically orienting the prenyl donor into the required position for optimal nucleophilic attack by LTrp. Indeed, inspection of these residues in the aligned sequences shows some degree of correlation between side chain identity and prenylation sites (Table 2). Table 2. Side Chains Proximal to the Prenyl Donor Moiety enzymes CymD, IlaO PriB, IptA MpnD, TleC SCO7467

prenylation site

capping group

residue 1

residue 2 residue 3

N C6 C7

Trp Tyr Tyr/Phe

Ala Leu Thr/Ser

Met Trp Met/Ala

Leu/Met Phe Ser/Ala

C5

Tyr

Leu

Trp

Phe

Specific Activity Assays. Whereas it was previously determined that CymD exclusively accepts DMAPP as the prenyl donor,26 its specificity for the prenyl acceptor had not been investigated. To screen potential substrates for CymD, a series of indoles and indole-like small molecules were evaluated by a coupled activity assay62 measuring the production of inorganic pyrophosphate (Figure 5). L-Trp, the native substrate of CymD, yielded a specific activity of 36.1 nmol of product (μmol of enzyme)−1 s−1. CymD also showed prenylation activity toward D-Trp, though at a rate roughly 20% of that measured with L-Trp. Indole, however, showed prenylation activity equal to that of L-Trp, demonstrating that the amino acid moiety of L-Trp is not critical for the proper orientation of the prenyl acceptor and that offset π−π stacking interactions alone are sufficient for catalysis. This is consistent with the mechanistic conclusions emanating from the crystallography results. Indoline and aniline showed very little activity, likely due to the increased nitrogen pKa [30.7 for aniline in dimethyl

Figure 5. Specific activities of CymD with DMAPP and various prenyl acceptor substrates. G


Article

Biochemistry Isothermal Titration Calorimetry (ITC). Thermodynamic measurements using ITC were performed to assess the binding of L-Trp and DMSPP to CymD (Table 3 and Figure S3). The

the indole nitrogen is optimally positioned for deprotonation by general base E64, followed by nucleophilic attack at the allylic cation intermediate resulting from departure of the diphosphate leaving group of DMAPP. Substrate L-Trp and cosubstrate analogue DMSPP are bound and oriented to direct exclusive N−C bond formation with the tertiary carbon of the dimethylallyl cation. Structural comparison of CymD to another bacterial indole prenyltransferase, PriB, provides additional insight into how each enzyme active site directs alternative regiochemistry for the electrophilic aromatic substitution reaction. While CymD and PriB both employ offset π−π interactions as the primary means of accommodating L-Trp with a common binding mode, differences in the residues proximal to the dimethylallyl moiety of DMSPP position the incipient carbocation intermediate adjacent to alternative ring atoms of the L-Trp indole group, thereby directing alternative prenylation reactions. The selectivity and cooperativity of substrate binding to CymD were studied by an activity assay and ITC, respectively. CymD showed prenylation activity toward only L-Trp, indole, and, to a lesser extent, D-Trp. Surprisingly, CymD showed no prenylation activity toward benzimidazole despite its lower nitrogen pKa, indicating that other factors must play important roles in the mechanism of electrophilic aromatic substitution. Finally, ITC measurements of binding of L-Trp and DMSPP to CymD suggest a synergistic interaction between the two substrates. Furthermore, the crystal structure of CymD reveals that both substrates may access the active site independently through different channels. L-Trp is a common precursor to myriad natural products, and the family of indole prenyltransferases constitutes a promising platform for the biosynthesis of such small molecules in biotechnology applications.1,15,16,59 As additional bacterial indole prenyltransferases are discovered and studied by structural methods, the correlation among the primary structure, active site architecture, and prenylation substrate specificity will be further clarified.

Table 3. Thermodynamic Values from ITC Measurements of L-Trp and DMSPP Binding to CymD

L-Trp

into CymD into CymD− DMSPP DMSPP into CymD DMSPP into CymD−L-Trp L-Trp

Kd (μM)

ΔH (kcal mol−1)

ΔS (cal mol−1 deg−1)

4.4 ± 0.4 1.1 ± 0.2

−10.6 ± 0.2 −16.0 ± 0.3

−11.0 −26.4

− 1.0 ± 0.2

− −5.2 ± 0.1

− 10.2

titration of L-Trp into CymD yielded a dissociation constant (Kd) of 4.4 μM, in good agreement with the Michaelis constant (KM) of 3.8 μM previously determined in steady-state kinetic assays.37 Notably, titration of L-Trp into CymD preincubated with DMSPP yielded a Kd of 1.1 μM, a 4-fold increase in affinity, suggesting a degree of synergy between L-Trp and DMSPP (or DMAPP) binding to CymD. The structural basis of this synergy is evident in the crystal structure of CymD. The simultaneous binding of L-Trp and DMSPP establishes a network of favorable offset π−π stacking interactions. Moreover, whereas the titration of DMSPP into CymD produced no measurable heats of binding, DMSPP titrated into CymD preincubated with L-Trp yielded a Kd of 1.0 μM, further demonstrating a synergistic interaction between the two substrates. The typical intracellular concentrations of tryptophan and DMAPP in bacteria are 12 and 140 μM, respectively.66,67 Inspection of the solvent-accessible surface of CymD reveals two channels accessing the active site cavity from two opposite sides of the protein surface (Figure 6), suggesting that each substrate can access the active site independently.

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CONCLUSIONS Reverse N-prenylation of L-Trp by CymD proceeds through the alkylation of the indole nitrogen, which is not ordinarily a nucleophilic atom in the biological or nonbiological context. To better understand the structural basis for this reaction, CymD was crystallized in the presence of L-Trp and DMSPP, an unreactive analogue of substrate DMAPP. The orientations of bound L-Trp and DMSPP in the ternary complex reveal how

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.9b00399. Sequence identity matrix for select bacterial indole prenyltransferases (Table S1), SDS−PAGE gel of

Figure 6. Solvent-accessible surface of CymD. Bound L-Trp and DMSPP are shown as yellow sticks. (Left) Channel accessing the L-Trp-binding pocket in the active site. (Right) Channel accessing the DMSPP-binding pocket in the active site. H


Article

Biochemistry

tobacco etch virus; Tris-HCl, tris(hydroxymethyl)aminomethane hydrochloride; TCEP-HCl, tris(2carboxyethyl)phosphine hydrochloride; PEG, polyethylene glycol.

purified CymD (Figure S1), sequence alignment of bacterial indole prenyltransferases (Figure S2), and isothermal titration calorimetry enthalpograms for LTrp and DMSPP binding to CymD (Figure S3) (PDF)

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Accession Codes

The atomic coordinates and crystallographic structure factors of native CymD, CymD complexed with L-Trp, and CymD complexed with L-Trp and DMSPP have been deposited in the Protein Data Bank as entries 6OS3, 6OS5, and 6OS6, respectively.

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REFERENCES

(1) Botta, B., Monache, G. D., Menendez, P., and Boffi, A. (2005) Novel prenyltransferase enzymes as a tool for flavonoid prenylation. Trends Pharmacol. Sci. 26, 606−608. (2) Li, S.-M. (2010) Prenylated indole derivatives from fungi: structure diversity, biological activities, biosynthesis and chemoenzymatic synthesis. Nat. Prod. Rep. 27, 57−78. (3) Winkelblech, J., Fan, A., and Li, S.-M. (2015) Prenyltransferases as key enzymes in primary and secondary metabolism. Appl. Microbiol. Biotechnol. 99, 7379−7397. (4) Sunassee, S. N., and Davies-Coleman, M. T. (2012) Cytotoxic and antioxidant marine prenylated quinones and hydroquinones. Nat. Prod. Rep. 29, 513−535. (5) Hao, Y., Pierce, E., Roe, D., Morita, M., McIntosh, J. A., Agarwal, V., Cheatham, T. E., Schmidt, E. W., and Nair, S. K. (2016) Molecular basis for the broad substrate selectivity of a peptide prenyltransferase. Proc. Natl. Acad. Sci. U. S. A. 113, 14037−14042. (6) Zverina, E. A., Lamphear, C. L., Wright, E. N., and Fierke, C. A. (2012) Recent advances in protein prenyltransferases: substrate identification, regulation, and disease interventions. Curr. Opin. Chem. Biol. 16, 544−552. (7) Xie, W., Zhou, C., and Huang, R. H. (2007) Structure of tRNA dimethylallyltransferase: RNA modification through a channel. J. Mol. Biol. 367, 872−81. (8) Tanner, M. E. (2015) Mechanistic studies on the indole prenyltransferases. Nat. Prod. Rep. 32, 88−101. (9) Bräuer, L., Brandt, W., Schulze, D., Zakharova, S., and Wessjohann, L. (2008) A structural model of the membrane-bound aromatic prenyltransferase UbiA from E. coli. ChemBioChem 9, 982− 992. (10) Li, S.-M. (2009) Evolution of aromatic prenyltransferases in the biosynthesis of indole derivatives. Phytochemistry 70, 1746−1757. (11) Kuzuyama, T., Noel, J. P., and Richard, S. B. (2005) Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products. Nature 435, 983−987. (12) Tello, M., Kuzuyama, T., Heide, L., Noel, J. P., and Richard, S. B. (2008) The ABBA family of aromatic prenyltransferases: broadening natural product diversity. Cell. Mol. Life Sci. 65, 1459− 1463. (13) Bonitz, T., Alva, V., Saleh, O., Lupas, A. N., and Heide, L. (2011) Evolutionary relationships of microbial aromatic prenyltransferases. PLoS One 6, No. e27336. (14) Christianson, D. W. (2017) Structural and chemical biology of terpenoid cyclases. Chem. Rev. 117, 11570−11648. (15) Fan, A., Winkelblech, J., and Li, S.-M. (2015) Impacts and perspectives of prenyltransferases of the DMATS superfamily for use in biotechnology. Appl. Microbiol. Biotechnol. 99, 7399−7415. (16) Qian, S., Clomburg, J. M., and Gonzalez, R. (2019) Engineering Escherichia coli as a platform for the in vivo synthesis of prenylated aromatics. Biotechnol. Bioeng. 116, 1116−1127. (17) Mai, P., Zocher, G., Ludwig, L., Stehle, T., and Li, S.-M. (2016) Actions of tryptophan prenyltransferases toward fumiquinazolines and their potential application for the generation of prenylated derivatives by combining chemical and chemoenzymatic syntheses. Adv. Synth. Catal. 358, 1639−1653. (18) Bartoli, G., Bencivenni, G., and Dalpozzo, R. (2010) Organocatalytic strategies for the asymmetric functionalization of indoles. Chem. Soc. Rev. 39, 4449−4465. (19) Bandini, M. (2013) Electrophilicity: The “dark-side” of indole chemistry. Org. Biomol. Chem. 11, 5206−5212. (20) Lakhdar, S., Westermaier, M., Terrier, F., Goumont, R., Boubaker, T., Ofial, A. R., and Mayr, H. (2006) Nucleophilic reactivities of indoles. J. Org. Chem. 71, 9088−9095.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: (215) 898-5714. ORCID

David W. Christianson: 0000-0002-0194-5212 Funding

This work was supported National Institutes of Health Grant GM56838. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Remote X-ray diffraction data collection was performed at Advanced Photon Source (APS) beamline 24-ID-C, a Northeastern Collaborative Access Team (NE-CAT) beamline funded by the National Institute of General Medical Sciences (NIGMS) from the National Institutes of Health (NIH) (P41 GM103403). The Pilatus 6M detector on beamline 24-ID-C is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Remote Xray diffraction data collection was also performed at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2, SLAC National Accelerator Laboratory, supported by the DOE Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIGMS (including Grant P41GM103393). X-ray diffraction data collection was also performed at Frontier Microfocusing Macromolecular Crystallography (FMX) beamline 17-ID-2 of National Synchrotron Light Source II (NSLS-II), a DOE Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract DE-SC0012704. The Life Science Biomedical Technology Research resource is primarily supported by the NIGMS through a Biomedical Technology Research Resource P41 grant (P41GM111244) and by the DOE Office of Biological and Environmental Research (KP1605010). The authors thank the following beamline scientists for their assistance with data collection: Dr. Narayanasami Sukumar (APS), Dr. Tzanko Doukov (SSRL), Dr. Wuxian Shi (NSLS-II), and Dr. Babak Andi (NSLS-II).

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ABBREVIATIONS DMAPP, dimethylallyl diphosphate; DMSPP, dimethylallyl Sthiolodiphosphate; PDB, Protein Data Bank; ITC, isothermal titration calorimetry; GFP, green fluorescent protein; TEV, I


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Biochemistry (21) Walsh, C. T. (2014) Biological matching of chemical reactivity: pairing indole nucleophilicity with electrophilic isoprenoids. ACS Chem. Biol. 9, 2718−2728. (22) Barry, S. M., Kers, J. A., Johnson, E. G., Song, L., Aston, P. R., Patel, B., Krasnoff, S. B., Crane, B. R., Gibson, D. M., Loria, R., and Challis, G. L. (2012) Cytochrome P450-catalyzed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis. Nat. Chem. Biol. 8, 814−816. (23) Zeng, J., and Zhan, J. (2011) Characterization of a tryptophan 6-halogenase from Streptomyces toxytricini. Biotechnol. Lett. 33, 1607− 1613. (24) Alkhalaf, L. M., and Ryan, K. S. (2015) Biosynthetic manipulation of tryptophan in bacteria: pathways and mechanisms. Chem. Biol. 22, 317−328. (25) Yu, X., Liu, Y., Xie, X., Zheng, X.-D., and Li, S.-M. (2012) Biochemical characterization of indole prenyltransferases: filling the last gap of prenylation positions by a 5-dimethylallyltryptophan synthase from Aspergillus clavatus. J. Biol. Chem. 287, 1371−1380. (26) Schultz, A. W., Lewis, C. A., Luzung, M. R., Baran, P. S., and Moore, B. S. (2010) Functional characterization of the cyclomarin/ cyclomarazine prenyltransferase CymD directs the biosynthesis of unnatural cyclic peptides. J. Nat. Prod. 73, 373−377. (27) Grundmann, A., Kuznetsova, T., Afiyatullov, S. S., and Li, S.-M. (2008) FtmPT2, an N-prenyltransferase from Aspergillus f umigatus, catalyses the last step in the biosynthesis of fumitremorgin B. ChemBioChem 9, 2059−2063. (28) Grundmann, A., and Li, S.-M. (2005) Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus f umigatus. Microbiology 151, 2199− 2207. (29) Parajuli, A., Kwak, D. H., Dalponte, L., Leikoski, N., Galica, T., Umeobika, U., Trembleau, L., Bent, A., Sivonen, K., Wahlsten, M., Wang, H., Rizzi, E., De Bellis, G., Naismith, J., Jaspars, M., Liu, X., Houssen, W., and Fewer, D. P. (2016) A unique tryptophan Cprenyltransferase from the kawaguchipeptin biosynthetic pathway. Angew. Chem., Int. Ed. 55, 3596−3599. (30) Yin, W.-B., Grundmann, A., Cheng, J., and Li, S.-M. (2009) Acetylaszonalenin biosynthesis in Neosartorya f ischeri. Identification of the biosynthetic gene cluster by genomic mining and functional proof of the genes by biochemical investigation. J. Biol. Chem. 284, 100−109. (31) Unsö ld, I. A., and Li, S.-M. (2005) Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus f umigatus. Microbiology 151, 1499− 1505. (32) Ozaki, T., Nishiyama, M., and Kuzuyama, T. (2013) Novel tryptophan metabolism by a potential gene cluster that is widely distributed among actinomycetes. J. Biol. Chem. 288, 9946−9956. (33) Elshahawi, S. I., Cao, H., Shaaban, K. A., Ponomareva, L. V., Subramanian, T., Farman, M. L., Spielmann, H. P., Phillips, G. N., Thorson, J. S., and Singh, S. (2017) Structure and specificity of a permissive bacterial C-prenyltransferase. Nat. Chem. Biol. 13, 366− 368. (34) Awakawa, T., Zhang, L., Wakimoto, T., Hoshino, S., Mori, T., Ito, T., Ishikawa, J., Tanner, M. E., and Abe, I. (2014) A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis. J. Am. Chem. Soc. 136, 9910−9913. (35) Kremer, A., Westrich, L., and Li, S.-M. (2007) A 7dimethylallyltryptophan synthase from Aspergillus f umigatus: overproduction, purification and biochemical characterization. Microbiology 153, 3409−3416. (36) Schultz, A. W., Oh, D.-C., Carney, J. R., Williamson, R. T., Udwary, D. W., Jensen, P. R., Gould, S. J., Fenical, W., and Moore, B. S. (2008) Biosynthesis and structures of cyclomarins and cyclomarazines, prenylated cyclic peptides of marine actinobacterial origin. J. Am. Chem. Soc. 130, 4507−4516. (37) Qian, Q., Schultz, A. W., Moore, B. S., and Tanner, M. E. (2012) Mechanistic studies on CymD: a tryptophan reverse Nprenyltransferase. Biochemistry 51, 7733−7739.

(38) Luk, L. Y. P., Qian, Q., and Tanner, M. E. (2011) A cope rearrangement in the reaction catalyzed by dimethylallyltryptophan synthase? J. Am. Chem. Soc. 133, 12342−12345. (39) Mahmoodi, N., and Tanner, M. E. (2013) Potential rearrangements in the reaction catalyzed by the indole prenyltransferase FtmPT1. ChemBioChem 14, 2029−2037. (40) Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R., and Leslie, A. G. W. (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67, 271−281. (41) Kabsch, W. (2010) XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 125−132. (42) Evans, P. R., and Murshudov, G. N. (2013) How good are my data and what is the resolution? Acta Crystallogr., Sect. D: Biol. Crystallogr. 69, 1204−1214. (43) Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67, 235−242. (44) McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658−674. (45) Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., de Beer, T. A. P., Rempfer, C., Bordoli, L., Lepore, R., and Schwede, T. (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296−W303. (46) Stein, N. (2008) CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641−643. (47) Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., GrosseKunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 213−221. (48) Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 486−501. (49) Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 12−21. (50) The PyMOL Molecular Graphics System, version 2.0, Schrödinger, LLC. (51) Krissinel, E., and Henrick, K. (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2256−2268. (52) Krissinel, E. (2012) Enhanced fold recognition using efficient short fragment clustering. J. Mol. Biochem. 1, 76−85. (53) Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774−797. (54) Bürgi, H. B., Dunitz, J. D., Lehn, J. M., and Wipff, G. (1974) Stereochemistry of reaction paths at carbonyl centres. Tetrahedron 30, 1563−1572. (55) Ma, J., Huang, H., Xie, Y., Liu, Z., Zhao, J., Zhang, C., Jia, Y., Zhang, Y., Zhang, H., Zhang, T., and Ju, J. (2017) Biosynthesis of ilamycins featuring unusual building blocks and engineered production of enhanced anti-tuberculosis agents. Nat. Commun. 8, 391. (56) Takahashi, S., Takagi, H., Toyoda, A., Uramoto, M., Nogawa, T., Ueki, M., Sakaki, Y., and Osada, H. (2010) Biochemical characterization of a novel indole prenyltransferase from Streptomyces sp. SN-593. J. Bacteriol. 192, 2839−2851. J


Article

Biochemistry (57) Ma, J., Zuo, D., Song, Y., Wang, B., Huang, H., Yao, Y., Li, W., Zhang, S., Zhang, C., and Ju, J. (2012) Characterization of a single gene cluster responsible for methylpendolmycin and pendolmycin biosynthesis in the deep sea bacterium Marinactinospora thermotolerans. ChemBioChem 13, 547−552. (58) Chojnacki, S., Cowley, A., Lee, J., Foix, A., and Lopez, R. (2017) Programmatic access to bioinformatics tools from EMBL-EBI update: 2017. Nucleic Acids Res. 45, W550−W553. (59) Mori, T., Zhang, L., Awakawa, T., Hoshino, S., Okada, M., Morita, H., and Abe, I. (2016) Manipulation of prenylation reactions by structure-based engineering of bacterial indolactam prenyltransferases. Nat. Commun. 7, 10849. (60) Metzger, U., Schall, C., Zocher, G., Unsöld, I., Stec, E., Li, S.M., Heide, L., and Stehle, T. (2009) The structure of dimethylallyl tryptophan synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria. Proc. Natl. Acad. Sci. U. S. A. 106, 14309−14314. (61) Schuller, J. M., Zocher, G., Liebhold, M., Xie, X., Stahl, M., Li, S.-M., and Stehle, T. (2012) Structure and catalytic mechanism of a cyclic dipeptide prenyltransferase with broad substrate promiscuity. J. Mol. Biol. 422, 87−99. (62) Upson, R. H., Haugland, R. P., Malekzadeh, M. N., and Haugland, R. P. (1996) A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal. Biochem. 243, 41−45. (63) Bordwell, F. G., and Algrim, D. J. (1988) Acidities of anilines in dimethyl sulfoxide solution. J. Am. Chem. Soc. 110, 2964−2968. (64) Bordwell, F. G. (1988) Equilibrium acidities in dimethyl sulfoxide solution. Acc. Chem. Res. 21, 456−463. (65) Bordwell, F. G., Bartmess, J. E., and Hautala, J. A. (1978) Alkyl effects on equilibrium acidities of carbon acids in protic and dipolar aprotic media and the gas phase. J. Org. Chem. 43, 3095−3101. (66) Milo, R., Jorgensen, P., Moran, U., Weber, G., and Springer, M. (2010) BioNumbers-the database of key numbers in molecular and cell biology. Nucleic Acids Res. 38, D750−D753. (67) Fisher, A. J., Rosenstiel, T. N., Shirk, M. C., and Fall, R. (2001) Nonradioactive assay for cellular dimethylallyl diphosphate. Anal. Biochem. 292, 272−279.

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Structural Basis of Tryptophan Reverse N-Prenylation Catalyzed by

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