ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 296, No. 1, July, pp. 308313,1992

Purification Tryptophan

and Characterization of Dimethylallyl Synthase from Claviceps Pwpurea’

John C. Gebler and C. Dale Poulter2 Department of Chemistry, University of Utah, Salt Lake City, Utah 841 I2

Received January

24, 1992, and in revised form March 16, 1992

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of L-tryptophan by dimethylallyl diphosphate to form 4-(y,y-dimethylallyl)-L-tryptophan. The enzyme from mycelia of Clavicepspurpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an a2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for KM in the metal-free EDTA buffer and V,, were determined (KkMApp ,14 PM; &&tryptophan, 40 PM; V,, ,2 15 nmol mine1 mg-l), 4 mM CaCl, (Kgmpp, 8.0 PM; Kktruptoph”“, 17 PM; V mBX, 504 nmol min-’ mg-l), and 4 mM MgC12 (KgMApp, 8.0 MM; #&tryptopban, 12 NM; V,,, 455 nmol min-’ mg-‘). The product was isolated and characterized by lH NMR, uv, and FAB mass spectrometry. 0 1s~ Academic PWS, IUC.

The fungus Clauiceps has a dark history due to the effects of ingesting ergot alkaloids found in infected rye and mixed grains (1). Overdoses lead to convulsions, often accompanied by hallucinations, and gangrene due to contraction of smooth muscles (2). The first pathway-specific step in the biosynthesis of ergot alkaloids is the alkylation of L-tryptophan at C(4) by dimethylallyl diphosphate (DMAPP)3 (3, 4) accompanied by inversion of configui This work was supported by National Institutes of Health Grant GM21328. * To whom correspondence should be addressed. 3 Abbreviations used: DMAPP, dimethylallyl diphosphate; DMAT, dimethylallyl tryptophan; ATCC, American Type Culture Collection; SDS, sodium dodecyl sulfate, PMSF, phenylmethylsulfonyl fluoride; FAB, fast atom bombardment; TFA, trifluoroacetic acid; PAGE, polyacrylamide gel electrophoresis. 308

ration at C(1) of DMAPP (5). The reaction is catalyzed by dimethylallyl tryptophan (DMAT) synthase, a prenyltransferase that uses the aromatic indole ring in tryptophan as a prenyl acceptor. Purifications of DMAT synthase from Claviceps sp. strain SD58 have been reported. Lee et al. described a monomeric enzyme with a molecular mass of 70-73 kDa (6), while Cress et al. determined that the enzyme was an a2 dimer containing 34-kDa subunits (7). The latter group reported that DMAPP and tryptophan each showed mixed cooperativity (negative to positive with increasing concentration). Unlike all other known prenyltransferases, DMAT synthase was active in the absence of divalent metal ions. However, Lee et al. found mild stimulation of activity upon addition of a variety of metals, including Ca2+ and Mg2+, while Cress et al. reported that Ca2+ was an allosteric effector which deregulated the enzyme at concentrations above 20 mM. During attempts to purify DMAT synthase from Clauiceps sp. for mechanistic studies, we encountered a loss of activity in our cultures. Attempts to reestablish a culture that produced reasonable levels of enzyme from those used previously (6,7) were unsuccessful. In addition, the enzyme was rather unstable, even when stored at -20°C. In a search for a more reliable source, we purified and characterized DMAT synthase from Claviceps purpurea, ATCC strain 26245. Enzyme from this source proved to be stable for prolonged periods at -20°C in buffers containing glycerol. EXPERIMENTAL

PROCEDURES

General. All enzyme isolation procedures were conducted at 4°C and in plasticware, where possible. Jar milling was accomplished with a Norton l-tier jar mill using porcelain jars. Whatman lOO- and 30-ml homogenizers were used for homogenizing protein pellets. Mixed hydrophobic/ion-exchange chromatography was conducted with Spectrum glass chromatography columns. HPLC chromatography was at 25’C. All chromatographic steps were monitored continuously at 280 nm. Centrifugal ultrafiltration was accomplished with Amicon Centricon 30 and Centriprep 30 microconcentrators (MW cutoff point 30,000). 0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. .

DIMETHYLALLYL

TRYPTOPHAN

A SCHEME

1

Dialysis was performed in Spectrapor dialysis bags (25.5 mm, MW cutoff 6000-8000 Da). All centrifugations were at a rotor temperature of 4°C. ‘H NMR spectra were recorded at 300 MHz and are reported in parts per million downfield from 3-(trimethylsilyl)-1-propane-sulfonic acid. All NMR spectra were obtained in deuterium oxide. Materials. C. purpurea (strain 26245) was obtained from the American Type Culture Collection (ATCC). DMAPP and [1-3H]DMAPP were synthesized by the method of Davisson et al. (8, 9). Elymoclavine for use as a standard in calorimetric determinations of alkaloid content was provided by Dr. Heinz Floss. Yeast extract and agar were purchased from Difco. Sucrose, mannitol, Tris, diethyldithiocarbamate, thioglycolate, glycerol, sodium dodecyl sulfate (SDS), L-tryptophan, 2-mercaptoethanol, and proteins for molecular weight and isoelectric focusing markers were purchased from Sigma. Ammonium sulfate (Ultra-Pure) was purchased from Schwarz/Mann. Sepharose 4B and Sephacryl S200 HR exclusion gels were from Pharmacia, acrylamide and bis-acrylamide (monomers) were from IBI, and Dowex AG 5OW-X4 analytical ion-exchange resin was from Bio-Rad. Polyampholytes for IEF were purchased from Serva. n-Butyl Sepharose was synthesized from CNBractivated Sepharose and n-butylamine. Deuterium oxide was purchased from Cambridge Isotope Laboratories. L-[3-“ClTryptophan (59 rCi/ pmol) was obtained from Amersham. Growth media for Clauiceps purpurea. Full-growth media, NL-406 (lo), contained mannitol(50.0 g/liter, 0.27 M), sucrose (50.0 g/liter, 0.15 M), yeast extract (3.0 g/liter), succinic acid (5.4 g/liter, 45 mM), KHzPOl (0.1 g/liter, 0.74 mM), MgSO, * 7H20 (0.3 g/liter, 1.2 mM), FeSO,. 7Hz0 (10.0 mg/liter, 0.036 mM), and ZnSOI* 7H20 (4.4 mg/liter, 0.015 mM). The mixture was stirred until the ingredients had dissolved, the pH was adjusted to 5.4 using concentrated NH,OH, and the solution was sterilized by autoclaving. Cultures for long-term storage. Agar slant cultures from ATCC were allowed to grow for 1 week at room temperature. When the entire agar surface was covered with growth, 2 ml of sterile 0.9% NaCl solution was added. The surface of the agar was gently rubbed with a sterile platinum wire to remove the fungus, while not disturbing the agar. Three seed cultures were prepared by addition of 0.67 ml of the Clauiceps solution to 25 ml of fresh growth media. The seed cultures were incubated with shaking for 10 days at 25°C to give a thick creamy off-white suspension. The contents of each culture were centrifuged, the supernatant was decanted, and the pellet was washed with 0.9% NaCl solution. The suspension was centrifuged, resuspended in 75 ml of 0.9% NaCl, and mixed in a blender with two 5-s bursts at low speed. Glycerol was added to produce a 10% (v/v) glycerol/water solution. Samples (2 ml) were placed in sterile plastic tubes and stored at -78°C. Frozen cultures were Cultures for purification of DMAT synthuse. used to inoculate four 250.ml Erlenmeyer flasks containing 50 ml of sterile full growth media. The flasks were stoppered with foam plugs and incubated with shaking for 5-7 days at 25°C. The mature seed cultures were washed with 0.9% NaCl solution as described above. The pellets were resuspended in 200 ml of 0.9% NaCl solution and mixed in a blender with two 5-s low speed bursts. Five milliliters of the seed culture mixture was added to 25-40 Roux culture bottles, each containing 200 ml of growth media. The bottles were stoppered with foam plugs and stored lying flat at 25°C until alkaloid production was detected by

SYNTHASE

309

a calorimetric assay (lo-14 days). Mature mycelia were collected by vacuum filtration and thoroughly washed with distilled water. The fungus was freeze-dried and stored in plastic freezer bags at -78°C until needed. A 2.0-ml sample of media Calorimetric determination of alkaloids. from an active stationary culture was spun for 10 min at 1520g. NH,OH solution (lo%, 100 ~1) and 2 ml of CHCls were agitated on a vortex mixer for 30 s and allowed to settle for 5 min. A l-ml portion of the CHCls layer was concentrated to dryness under a stream of dry nitrogen. To the residue were added 1 ml of 2% succinic acid solution and 2 ml of Van Urk reagent [35 ml H20, 65 ml concentrated sulfuric acid, 0.15 ml 10% (w/v) ferric chloride solution, and 200 mg (1.34 mmol) 4-(dimethylamino)-benzaldehyde] (11). The materials were mixed and allowed to stand for 20 min. Absorption at 580 nm was measured against a blank of water containing Van Urk reagent, and alkaloid concentrations (pg/ ml) were determined from a standard curve using measured amounts of elymoclavine. Twenty-eight Determination of dry cell weight and enzyme actiuity. 125.ml Erlenmeyer flasks containing 25 ml of growth media were each inoculated with 1 ml of mature seed culture. Every 24 h, the contents of two flasks were filtered through tared 70-mm circles of filter paper. The filtrate was collected for a calorimetric assay to determine alkaloid content. The mycelial mats were washed with deionized water, lyophilized on the supporting filter paper, and weighed to determine dry cell weight. The mycelia were scraped from the filter papers, crushed, and placed in centrifuge tubes containing 4 ml of buffer (10 mM Tris-HCl, 20 mM 2mercaptoethanol, and 10% (v/v) glycerol, pH 8). The material was disrupted by sonication with a sufficient interval between bursts to ensure that the temperature did not rise above 10°C. The samples were then spun for 30 min at 18,OOOg,and enzyme activity was determined in the resulting supernatant. DMAT synthase assay. The final composition of the assay buffer was 500 PM L-tryptophan, 500 PM [3H]DMAPP (2 &i/pmol), 4 mM CaClz, 50 mM Tris-HCl, 20 mM 2-mercaptoethanol, and 10% glycerol (v/v), pH 7.8. Incubations were carried out for 10 min at 3O”C, and the reaction was stopped by the addition of 3 ml of distilled water at 4°C. The incubation mixture was transferred to a lo-cc syringe barrel containing 3 ml of cation-exchange resin (H+ form) and washed three times with 10 ml of distilled water. The product was eluted into a 15.ml glass scintillation vial with 5 ml NH40H:Hz0:CH30H (2:3:5). The vials were placed in a dry block heater (9O”C), and solvent was removed under a stream of nitrogen. To the residue was added 1 ml of distilled water and 10 ml of OptiFluor scintillation cocktail. The contents of the vials were thoroughly mixed, and radioactivity was determined by liquid scintillation spectrometry. Lyophilized mycelia (80 g) and 5 Purification of DMAT synthuse. mg of PMSF were ground in a ball mill for 45 min. A 1300-ml portion of extraction buffer (20 mM 2-mercaptoethanol, 20 mM Tris-HCl, 1 mM PMSF, 20 mM thioglycolate, 20 mM diethyldithiocarbamate, 20 mM CaC12, and 10% (v/v) glycerol, pH 7.8) was added, and milling was continued for an additional 15 min. The suspension was spun for 30 min at 7000g. The resulting supernatant was collected and treated with Cell Debris Remover (Whatman; 80 mg/ml). The mixture was gently mixed with a Teflon rod for 5 min and then filtered through coarse (Whatman 41) filter paper. The filtrate was brought to 35% (w/v) saturation with (NH&SO, and spun at ll,OOOg for 30 min. The supernatant was taken to 55% (w/v) saturation with (NH&SO, and spun again for 30 min at 11,OOOg.The 35-55% pellet was dissolved in chromatography buffer (50 mM Tris-HCl, 20 mM 2-mercaptoethanol, 10% glycerol (v/ v), pH 7.8) and dialyzed for 12 h against 2 liters of the same buffer with one change after 4 h. A 5 X 15-cm n-butyl Sepharose column was equilibrated with chromatography buffer. Dialyzed protein was clarified by centrifugation at 150,OOOgand applied to the column at a rate of 3.0 ml/min. The column was then washed with buffer until the absorbance of the effluent fell below 0.1. Protein was eluted at 3.4 ml/min with a 1200-ml linear gradient of 0 to 300 mM KC1 in chromatography buffer. Fractions of 13 ml were collected, and those containing DMAT synthase activity were pooled.

310

GEBLER

AND

The protein was precipitated with (NH,)sSOI (55% (w/v) saturation). The suspension was spun for 30 min at 17,000g. The pellet was dissolved in salt-free chromatography buffer and dialyzed against 2 liters of the same buffer. The dialyzed protein was applied to a 10 X 60-mm Q Sepharose column. The column was washed with chromatography buffer and eluted at 1.0 ml/min with a 200-ml linear gradient of 0 to 0.5 M KCl. Fractions (5 ml) were collected, and those containing DMAT synthase activity were combined and concentrated by ultrafiltration. The concentrate from Q Sepharose chromatography was taken to 1 M (NH&SO, with solid (NH,)sSOI and applied to a 10 X lOO-mm Phenyl Superose column equilibrated with chromatography buffer in 1 M (NH&SOI. The column was washed with the same buffer, and protein was eluted at 0.65 ml/min with a decreasing linear gradient of 1.0 to 0.7 M (NH,)sSOI in 35 ml, followed by 0.7 to 0 M (NH&SO, in 120 ml. Fractions of 4 ml were collected. Those with DMAT synthase activity were combined and the concentrate was desalted by ultrafiltration. Dialyzed protein from hydrophobic chromatography was applied to a 7.5 X 75-mm Protein Pak (Waters) column at a flow rate of 0.75 ml/ min. The column was washed with chromatography buffer and eluted at 0.75 ml/min with a 120-ml linear gradient of 0 to 300 mM KC1 in chromatography buffer. Active fractions were combined, concentrated by ultrafiltration, suspended in storage buffer (50 mM Tris-HCl, 20 mM 2-mercaptoethanol, 50% v/v glycerol, pH 7.8) at a concentration of 2 mg/ml, and stored at -20°C. Molecular weight determination by gel exclusion chromatography. A 1.5 X 87.5-cm column of Sephacryl S-200 superfine was equilibrated with chromatography buffer containing 0.1 M KCl. The flow rate was adjusted to 2 ml/h and the column was calibrated with egg albumin (45 kDa), bovine serum albumin (67 kDa), and rabbit muscle aldolase (160 kDa). DMAT synthase (82.5 rg) was added to the standards and applied onto the column. Elution of protein was monitored at 280 nm, and fractions (2 ml) were collected and assayed for DMAT synthase activity. Enzymatic synthesis of dimethylallyl tryptophan. To a 15-ml glass vial with a screw cap were added DMAPP (0.5 ml; 11 mM), L-tryptophan (100 @k 100 mM), assay buffer containing CaCls (250 pl; 200 mM), and DMAT synthetase (82.5 pg). The mixture was incubated at 30°C for 24 h. The incubation mixture was filtered through a microconcentrator (MW cutoff 10,000). The filtrate was collected, and DMAT was purified by reverse phase chromatography on a Walters Radial-Pak Cls column upon elution with a linear gradient of 0 to 50% acetonitrile in 50 mM sodium phosphate, pH 4.3. Fractions containing DMAT were combined and lyophilized. The white residue was dissolved in deuterium oxide and lyophilized in succession four times. The residue was then dissolved in D20 (99.996%); ‘H NMR (300 MHz) 6 1.76 (3H, s, allylic CH,), 1.79 (3H, s, allylic CHs), 3.25 (lH, dd, J = 15.6 Hz, J = 10.4 Hz, H at C(p)), 3.71 (lH, dd, J = 15.6 Hz, J = 4.5 Hz, H at C(p)), 3.79 (2H, m, H’s at C(l”)), 3.89 (lH, dd, J = 10.4 Hz, J = 4.5 Hz, H at C(a)), 5.37 (lH, m, H at C(2’)), 6.99 (lH, d, J = 7.5 Hz, H at indole C(5)), 7.20 (lH, dd, J = 8.1 Hz, J = 7.5 Hz, H at indole (C(6)), 7.30 (lH, s, H at indole C(2)), and 7.41 (lH, d, J = 8.1 Hz, H at indole C(7)), mass spectrum (FAB in 0.1% TFA and glycerol), m/z (rel intensity) 115 (100) [TFA], 185 (32.9) [glycerol], 207 (9.3), 243 (48.6), 244.1 (1.8), 273 (1.6) [M + 11; uv (HzO) X,, at 269 nm (%I,, 6605).

POULTER

on an agar medium (12). We found this problem was overcome with C. purpurea by storing the organism at -7&Y% and using the frozen cultures to inoculate fresh media for large-scale fermentations. It was previously noted that 2- to 3-week-old stationary cultures were the best source of enzyme and that adequate levels of DMAT synthase were present when the culture media turned a deep wine red (7). We found substantial variations in enzyme activity using this method to determine when to harvest and decided to examine the correlation between the specific activity of DMAT synthase and the rate of alkaloid production. The concentration of alkaloids in the media was easily measured with a colorimetric assay based on the Van Urk reagent (6, 13). This reagent is sensitive toward indole alkaloids and produces a violet color, whose intensity correlates linearly with alkaloid concentration. Measurements of absorbance at 580 nm were compared to a standard curve determined with elymoclavine to determine the concentration of indole alkaloids in the media of Clauiceps cultures. Determinations of dry cell weight, alkaloid concentration, and DMAT synthase activity indicated that optimal specific activities of the enzyme were obtained when the rate of alkaloid production was at a maximum. This varied from 12 to 16 days after inoculation. The cultures were assayed daily, and mycelia were harvested when alkaloid production was maximal. The material was dried and stored at -78°C until needed. The first steps in our Purification of DMAT synthme. purification of DMAT synthase from C. purpurea, summarized in Table I, were based on the procedure reported by Cress et al. for the enzyme from Claviceps sp. (7). However, we experienced difficulties with reproducibility, including rather rapid loss of activity upon standing. The specific activity of crude homogenates increased from 0.9 to 4 nmol mine1 mg-l when PMSF was added directly to lyophilized mycelia before grinding in the ball mill. Treatment with the protease inhibitor also improved the stability of the enzyme throughout the purification. TABLE

I

Isolation of DMAT Synthase from 80 g of Lyophilized Mycelia from Claviceps purpurea Overall

Protein RESULTS

AND

DISCUSSION

Physiological studies. In preliminary studies, we obtained cultures of Clauiceps sp. (strain SD58), which produced DMAT synthase. However, upon storage of the original stock on agar slants, fresh cultures lost their ability to produce the enzyme and attempts to reestablish activity from the original sources were unsuccessful. Floss et al. reported that the level of alkaloid production in Claviceps decreased by up to 100-fold after nine transfers

Purification

step

pH 7.8 supernatant CDR-treated supernatant 35-55% (NH,)2S0, n-Butyl Sepharose Q Sepharose Fast Flow Phenyl Superose Protein Pak

Units” 13.1 12.5 7.6 4.5 3.1 2.2 1.0

’ Units in pm01 mini. * Specific activity in pm01 min-’

yield

Purification

bxd

S.A.”

(%)

(fold)

3225 2760 600 141 45 26 2

0.004 0.005 0.013 0.032 0.07 0.10 0.50

100 95 58 34 24 16 8

-

mg-‘.

1.3 3 8 18 25 125

DIMETHYLALLYL

TRYPTOPHAN

The original protocol used precipitation with (NH&SO4 to concentrate fractions containing DMAT synthase between chromatographic steps. In our hands, high concentrations of the salt had an inhibitory effect that was not fully restored upon dialysis. After the nbutyl Sepharose step, concentrations were done by ultrafiltration using a membrane with a 30-kDa retention. This procedure allowed us to concentrate the enzyme rapidly without loss of activity. Following chromatography on Protein Pak, the specific activity of the enzyme was 0.5 pmol min-’ mg-‘. DMAT synthase purified in this manner was active for more than 6 months when stored at -20°C in glycerol-containing buffers. The purified protein had a p1 at 6.0 and a pH optimum (V,,,) at 7.5. In a preparative-scale incubation, L-tryptophan and DMAPP were incubated with DMAT synthase at 30°C for 24 h. During the course of the incubation, a white precipitate, presumably calcium pyrophosphate, formed. The water-soluble product was separated from enzyme and the precipitate by filtration, concentrated, and purified by HPLC. The material was then characterized by uv spectroscopy, FAB mass spectrometry, and ‘H NMR spectroscopy. The molecular mass of native DMAT synthase was 105 kDa, as measured by gel exclusion chromatography. SDSPAGE gave a single band at 53 kDa (6). These results suggest that the enzyme is a homodimer (7). Our results differ from those reported for DMAT synthase from Clauiceps sp. Lee et al. (6) reported a monomer with a molecular mass of 70-73 kDa, while Cress et al. (7) concluded the enzyme was a 70-kDa homodimer. In experiments for batches of enzyme without PMSF in the extraction buffer, we saw heterogeneity in SDS gels of purified protein. Proteolysis may be responsible for the discrepancies in molecular mass. Effect of metal ions. Most prenyltransferases require a divalent metal ion for activity (14). In the case of farnesyl diphosphate synthase, the monomagnesium salts of isopentenyl diphosphate and geranyl diphosphate are the true substrates (15). For the farnesyl diphosphate:protein

311

SYNTHASE TABLE

II

Kinetic Constants for 1’-Aryl Condensation between L-Tryptophan and DMAPP

Metal ion

KgMAPP ( jtM)

KFrypmh.+n cpM)

V me.x ( pm01 min-’ mg-‘)

14 z!z3 8k3 8fl

40 t 6 17 k 2 12 * 2

0.22 * 0.02 0.50 f 0.03 0.46 k 0.03

None

4 mM Ca2+ 4 mM Mg2+

transferase from rat brain, zinc is also required for activity (16). The only known exception is DMAT synthase, which is active in the presence of EDTA (7). Cress et al. also found that the enzyme is stimulated more than twofold in the presence of Ca2+ and Mg2+. In addition, Lee et al. reported that other metals such as Na+, Li+ Cu2+, and Fe2+ were mildly stimulatory (6). The effect of Ca2+ and Mg2+ on the activity of DMAT synthase from C. purpurea is shown in Fig. 1. V,,, versus concentration profiles are similar for both metals. V,,, was stimulated more than twofold at l-10 mM and decreased slowly at higher concentrations. At higher levels of Ca2+, the assay solutions became noticeably turbid, and precipitation of substrate may have contributed to the decrease in V,,,,,. Kinetic studies. Initial velocities were measured at less than 10% conversion in the linear range for the enzyme, and the kinetic constants for DMAT synthase are listed in Table II. Either Ca2+ or Mg2+ gave approximately twofold stimulations of V,,, with a concomitant two- to threefold reduction in KM’s for DMAPP and tryptophan at 4 mM metal ion. Under conditions of 4 mM metal or at high concentrations of substrates (5-l-O times KM), the enzyme showed typical Michaelis-Menten kinetics. However, convex plots, indicative of negative cooperativity, were seen at lower substrate concentrations in metalfree buffers (see Fig. 2). The magnitude of curvature was inversely proportional to substrate concentration. In the presence of 4 mM ca2+, the enzyme obeyed MichalisMenten kinetics (see Fig. 3), suggesting the loss of cooperativity. Cress et al. reported a similar phenomenon for the Claviceps sp. enzyme at 20 mM Ca2+ and proposed that the metal is a positive allosteric effector which deregulates the enzyme (7). CONCLUSIONS

10

20 Concentration

30

40

50

(mM)

FIG. 1. Effect of Cazf (A) and Mg2+ (0) on the velocity DMAT synthase.

(V,,,,,) of

C. purpurea is an excellent source of dimethylallyl tryptophan synthase. Frozen cultures of the fungus can be stored for extended periods without attention and used to inoculate fermentations that reproducibly yield the enzyme. The optimal time to harvest cultures varies from 12 to 16 days but can be ascertained by measuring the rate of accumulation of indole alkaloids in the media using a calorimetric assay based on the Van Urk reagent (13).

GEBLER AND POULTER

312

The specific activity of DMAT synthase in crude homogenates of mycelia from maximally producing cultures is normally 4 X lop3 pm01 min-l mg-l. DMAT synthase is subject to proteolysis during the early stages of purification. To prevent degradation, we included PMSF in the initial extraction buffer. The enzyme was purified to >90% homogeneity in six steps with an overall yield of 8% to give protein with a specific activity of 0.5 pm01 min-l mg- ‘. DMAT synthase is the only prenyltransferase that does not require a divalent metal ion for catalysis. The activity of the enzyme is, however, stimulated by Ca2+ and M2+, with maximal activity at metal ion concentrations from 4 to 10 mM. In the absence of metal ion and at substrate concentrations near KIM for DMAPP and tryptophan, double reciprocal plots of velocity versus substrate concentration are convex, indicative of negative cooperativity. The plots become linear at high substrate concentrations or upon addition of Ca2+. The metal appears to be a positive allosteric effector that deregulates the enzyme. The mechanism of action of DMAT synthase has not been established. It is generally assumed that all prenyltransferases catalyze electrophilic alkylations, including DMAT synthase (5), although this mechanism has been

o.oa- A 0.063

0.04-

0.02-

0.00 -0.x)

I 0.00

I

I

0.x)

0.20

lAtrp1 o.oa-

6

o.oa-

l/[DMAPPI FIG. 3. Double reciprocal plots of initial velocities versus substrate concentration in the presence of 4 mM Cax+. Fixed substrate concentrations were 5, 7, 10, and 20 pM for DMAPP (A); and 25, 34, 50, and (B). 100 pM for L-tryptophan

o.oo.*, 0.00

0.02

0.01

0.03

0.04

lAtrp1

established only for farnesyl &phosphate synthase (17). A similar reaction for DMAT synthase would be an example of an electrophillic aromatic substitution, a common reaction in organic chemistry. There are several other prenyltransferases involved in the biosynthesis of compounds such as ubiquinone (18), umbelliferone (19), echinulin (20), and clehydrotremetone (21) that also catalyze alkylation of aromatic rings, presumably by the same mechanism. It should be possible to address this issue in studies similar to those reported for farnesyl &phosphate synthase using alternate substrates whose reactivity can be predictably altered by substitution (17, 22). ACKNOWLEDGMENTS We thank Professor Hans Rilling for helpful discussions. This work was supported by NIH Grant GM21328.

0.00 , , , 0.05 0.00

,

,

,

0.10

, 0.15

*

, 0.20

MDMAPPI . ^ . . . . Double reciprocal plots ot imtial velocities versus substrate

FIG. 2. concentration in the presence of 2 mM EDTA. trations were 5, 7, 10, and 20 pM for DMAPP 100 PM for L-tryptophan (B).

Fixed substrate concen(A); and 25, 34, 50, and

REFERENCES 1. Christensen, C. W. (1975) Molds, Mushrooms, Univ. of Minnesota Press, Minneapolis.

and Mycotoxins,

2. Barger, G. (1931) Ergot and Ergotism, Gurney & Jackson, London. 3. Floss, H. G. (1976) Tetrahedron 32, 873-912. 4. Robbers, J. E. (1984) Ada Biotechnol.

Processes 3, 200-239.

DIMETHYLALLYL

TRYPTOPHAN

5. Shibuya, M., Chou, H. M., Fountoulakis, M., Hassam, S., Kim, S. Il., Kobaayashi, K., Otsuka, H., Rogalska, E., Cassady, J. M., and Floss, H. G. (1990) J. Am. Chem. Sot. 112, 297-304. 6. Lee, S.-L., Floss, H. G., and Heinstein, P. (1976) Arch. Biochem. Biophys. 177,84-94. 7. Cress, W. A., Chayet, L. T., and Rilling, H. C. (1981) J. Biol. Chem. 256, 10,917-10,923. 8. Davisson, V. J., Woodside, A. B., Stremler, K. E., Neal, T. R., Muehlbacher, M., and Poulter, C. D. (1986) J. Org. Chem. 51,4768-

4779. 9. Davisson, V. J., Zabriskie, T. M., and Poulter, C. D. (1986) Bioorg. Chem. 14,46-54. 10. Floss, H. G., and Groger, D. (1963) Einbauuersuche, 519. 11. Allport, N. L., and Cocking, T. T. Q. (1932) J. Phurm. Pharmacol. 5,341-346. 12. Floss, H. G., Hornemann, K. M., Jindra, A., Robbers, J. E., and Robertson, L. W. (1972) J. Bacterial. 112, 791-796. 13. Krupinski, V., Robbers, J. E., and Floss, H. G. (1976) J. Bacterial.

116,158-165.

313

SYNTHASE

14. Sagami, H., Ogura, K., Weiner, A., and Poulter, C. D. (1984) Biochem. Znt. k&661-667. 15. Laskovics, 1901.

F. M., and Poulter, C. D. (1981) Biochemistry

20,1893-

16. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J., and Brown, M. S. (1990) Cell 62, 81-88. 17. Poulter, C. D., and Rilling, H. C. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W., and Spurgeon, S. L., Eds.), Vol. 1, pp. 162-224, Wiley, New York. 18. Pennock, J. F., and Threlfall, D. R. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W., and Spurgeon, S. L., Eds.), Vol. 2, pp. 191-304, Wiley, New York. 19. Ellis, B. E., and Brown, S. A. (1974) Can. J. Biochem. 53,734-738. 20. Allen, C. M. (1972) Biochemistry 21. Lin, J. J., Ramstad, 13, 1809-1815. 22. Poulter,

11, 2154-2160.

E., and Heinstein,

P. (1974) Photochemistry

C. D., Wiggins, P. L., and Lee, A. T. (1981) J. Am. Chem.

Soc.103,3926-3927.