©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Aspen Isoprene Synthase, an Enzyme Responsible for Leaf Isoprene Emission to the Atmosphere (*)

Gary M. Silver (§) , Ray Fall (¶)

From the (1) Department of Chemistry and Biochemistry and the Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado 80309-0215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon emitted from many plant species to the atmosphere, where it plays an important role in atmospheric chemistry. An enzyme extracted from aspen (Populus tremuloides) leaves was previously found to catalyze the Mg-dependent elimination of pyrophosphate from dimethylallyl diphosphate (DMAPP) to form isoprene (Silver, G. M., and Fall, R.(1991) Plant Physiol. 97, 1588-1591). This enzyme, isoprene synthase, has now been purified 4000-fold to near homogeneity. The enzyme had a native molecular mass of 98-137 kDa and isoelectric point of 4.7 and contained 58- and 62-kDa subunits, implying that it is a heterodimer. Partial amino acid sequences of the two subunits indicated they are closely related to each other and that they do not share a strong homology with any other reported proteins. The isoprene synthase reaction was dependent on Mg or Mn, and the reaction products were shown to be isoprene and pyrophosphate with a stoichiometry close to 1:1. The K for DMAPP was high at 8 mM, and the k of 1.7 s was low, but similar to those of other allylic diphosphate-utilizing enzymes. It is argued that the isoprene synthase reaction may be much more efficient in vivo, where it is under light-dependent control. It seems probable that this unique enzyme, rather than non-enzymatic reactions, can account for the emission of hundreds of millions of metric tons of isoprene from plants to the global atmosphere each year.


INTRODUCTION

Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon emitted from many species of plants to the atmosphere, where it plays an important role in atmospheric chemistry (1, 2) . Although plant isoprene emission was first described nearly 40 years ago, very little is known about the biochemistry of isoprene production. Because isoprene is the simplest member of the isoprenoid family, it seems likely that it could be derived from the same C intermediates, isopentenyl diphosphate (IPP)() or dimethylallyl diphosphate (DMAPP), that give rise to the larger members of this family (e.g. monoterpenes, sterols, carotenes, rubber, etc.).

Isoprene emissions from plants are light and temperature-dependent and increase dramatically during leaf development (3, 4, 5) . It has been suggested that isoprene is produced non-enzymatically from DMAPP located in the thylakoid lumen (3) , but there is no direct evidence for this idea. Alternatively, on mechanistic grounds it seemed likely that an enzymatic reaction would be possible, where DMAPP could give rise to isoprene by an elimination-rearrangement reaction (see Fig. S1). We recently reported that such an enzyme does exist in aspen leaves (6) , and it has since been detected in leaves of other isoprene-emitting plants, including velvet bean (5) , and willow and the aquatic fern Azolla,() but, so far, not in non-emitting plants. The enzymatic reaction was found to be dependent on the presence of magnesium ion and was optimal at pH 8. These optimal conditions are similar to those found inside the chloroplast stroma during photosynthesis (7, 8) , suggesting that this enzyme, if located within the chloroplast, might be activated by light-dependent changes in chloroplast pH and Mg concentration. The temperature dependence of leaf isoprene emission, known since the 1960s (9) , parallels the temperature dependence of the enzymatic reaction, suggesting that plant isoprene emission is ultimately controlled by this enzyme's activity (10) .


Figure S1: Scheme 1



In order to further study this novel enzyme, it was necessary to purify it to near homogeneity. Many different techniques were tried in isolating this enzyme, and presented here are the methods that yielded the highest degree of enzyme stability, purification, and recovery. In addition, it was desirable to determine the protein's partial amino acid sequence and to measure its biochemical properties, allowing us to directly compare it with other enzymes that use prenyl diphosphate substrates.


EXPERIMENTAL PROCEDURES

Plant Materials and Reagents

Quaking aspen leaves (Populus tremuloides Michx.) were collected from various locations throughout the city of Boulder, CO. DMAPP was synthesized and purified according to the method of Davisson et al. (11) with reagents supplied by Aldrich (Milwaukee, WI). Verification of the structure and purity of the DMAPP was by thin-layer chromatography and by H and P NMR using a 300-MHz Varian VXR-300S instrument. Stock solutions of DMAPP were made up in 2 mM ammonium bicarbonate (pH 7) and stored at -20 °C. Polyclar AT (polyvinylpolypyrrolidone) was supplied by Serva Biochemicals (Westbury, NY). All other reagents were reagent grade.

Isoprene Synthase Assay

Isoprene production was assayed by gas chromatography with a mercuric oxide detector (GC/HgO) as described previously (6) , except that samples were incubated at 32 °C. Assay reaction mixtures typically contained 15 µl of enzyme, 12 mM MgCl, and 10 mM DMAPP and were incubated for varying times, typically 10 min, before headspace analysis. Although the DMAPP concentration was only slightly above the K, isoprene production was linear with respect to time and enzyme concentration. When necessary, isoprene synthase was diluted in buffer IPB, which consisted of 50 mM Tris-HCl (pH 8.0), 20 mM MgCl, 1% (v/v) polyethylene glycol 400 (PEG 400), and 2 mM dithiothreitol (DTT), supplemented with 5 mg/ml bovine serum albumin to stabilize the enzyme.

Substrate and Product Quantification

DMAPP disappearance in reaction mixtures was measured by HPLC using a C-18 reverse-phase column (Vydak, Hesperia, CA) equilibrated with 10:90 (v/v) acetonitrile:5 mM tetrabutylammonium phosphate (pH 7.5). Upon injection, a gradient was immediately started to 90:10 (v/v) acetonitrile:5 mM tetrabutylammonium phosphate (pH 7.5) over 8 min at 1 ml/min. These conditions were held for an additional 7 min. The column effluent was monitored at 202 nm. Authentic DMAPP (Sigma) was used to verify the retention time and concentration of DMAPP.

Inorganic pyrophosphate was assayed by the method of Justesen (12), except that NADPH production was monitored by fluorescence, instead of absorption at 340 nm, with a 5-10-fold increase in sensitivity (13) .

Allylic alcohol production was assayed by gas chromatography and HPLC. 3-Methyl-2-buten-1-ol (dimethylallyl alcohol) and 2-methyl-3-buten-2-ol (tertiary analog of dimethylallyl alcohol) standards were supplied by Aldrich. One-ml headspace samples from isoprene synthase reactions were assayed for isoprene and alcohol production using a Hewlett-Packard 5890 GC with flame ionization detection (GC/FID) and a 5% phenylmethyl silicone capillary column (0.31 mm inner diameter 25 m, Hewlett-Packard), run isothermally at 30 °C. Alcohols in the aqueous phase of reaction mixtures were measured by HPLC; 10-microliter aliquots were separated using a C-18 reverse-phase column and 20% (v/v) acetonitrile in water at a flow rate of 1 ml/min as the mobile phase with detection at 202 nm.

Purification of Isoprene Synthase

Unless otherwise indicated, all extraction and purification procedures were performed at 0-4 °C. Mature aspen leaves (100 g) were homogenized with a mortar and pestle with 1 liter of plant extract buffer (PEB; 50 mM Tris-HCl (pH 8.0), 20 mM MgCl, 5% glycerol, 2 mM DTT), supplemented with 20 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine HCl, and 100 g of Polyclar AT as suggested by Gegenheimer (14) . The extract was filtered through two layers of cheesecloth and a single layer of Miracloth (Calbiochem, La Jolla, CA) and centrifuged for 20 min at 12,000 g. The resulting supernatant was then centrifuged for 40 min at 40,000 g, yielding a final supernatant that was retained. Isoprene synthase activity was precipitated from the supernatant between 40 and 55% saturation with ammonium sulfate. The precipitate was collected by centrifugation, resuspended in 0.1 volume of PEB (approximately 100 ml) and dialyzed against two changes of 1 liter of PEB for 3 h and clarified using Nalgene (Rochester, NY) 0.45-µm vacuum filters. The protein was loaded slowly and incrementally onto a HiLoad Q-Sepharose 16/10 fast flow column (Pharmacia Biotech Inc.) using a 50-ml Pharmacia Superloop. After each incremental loading, the column was washed with PEB until the A of the eluant came back to near zero. Protein was eluted with a linear gradient from 0 to 1 M NaCl in PEB at 2 ml/min over 100 min. The most active fractions were pooled and concentrated to 3-5 ml using ultrafiltration. A 10 ml Superloop was used to load concentrated protein (0.5 to 1.5 ml) onto a Superdex 200 HR 16/60 gel filtration column (Pharmacia), equilibrated with buffer IPB. In addition, 100 mM NaCl was added to the column buffer to prevent tailing of the isoprene synthase peak. The column was run at 1 ml/min, and the most active fractions were pooled and concentrated to 0.5-1.5 ml using Microsep 30-kDa centrifugal concentrators (Filtron) and loaded onto a 5-ml Econo-Pac HTP (hydroxyapatite) cartridge (Bio-Rad), equilibrated with 10 mM sodium phosphate (pH 7.0), 1% (v/v) PEG 400, and 2 mM DTT. The column was washed with this buffer at 0.5 ml/min until the A returned to base line and eluted with a linear gradient from 10 to 200 mM sodium phosphate (pH 7.0), 1% (v/v) PEG 400, and 2 mM DTT over 80 min. The most active fractions were pooled and concentrated down to 20-30 µl using a Microcon-3 centrifugal concentrator (Amicon, Beverly, MA) and then resuspended in 200 µl of IPB. One-hundred µl of this preparation were then loaded onto a Bio-Sil SEC 250-5 size exclusion chromatography column (Bio-Rad) and resolved by HPLC at room temperature, equilibrated, and run at 1 ml/min with buffer IPB kept chilled on ice. The most active fractions were pooled and concentrated to 50-100 µl using a centrifugal concentrator and stored at -70 °C.

As an analytical step, some preparations of isoprene synthase were run on a Mono S HR 5/5 cation exchange column (Pharmacia). This separation was performed at room temperature using HPLC. After 50 µl of sample were loaded, the column was washed for 6 min with buffer IPB at 0.5 ml/min and eluted with a linear gradient from 0 to 0.5 M NaCl in IPB over 9 min. Fractions were collected in tubes placed on ice. The highly active fractions were concentrated to 30 µl using a Microcon-3 centrifugal concentrator and stored at -70 °C. Protein concentrations at each step were measured using Bradford reagent (15) .

Native Molecular Weight Determinations

The native molecular weight of isoprene synthase was estimated by comparing the elution volume of the enzyme from the Superdex 200 and Bio-Sil SEC 250 columns to gel filtration standards (1.35-670 kDa, Bio-Rad). Sodium chloride (0.1 M) was included in the Superdex 200 column buffer to prevent peak tailing. Analysis of Proteins by SDS-PAGE-Enzyme fractions were analyzed by denaturing SDS-PAGE using 1.0-mm-thick, 8-16% gradient precast Novex mini-gels. The Novex Tris-glycine SDS sample buffer was supplemented with 1 mM thioglycolate, 1 mM sodium EDTA, and 100 mM DTT. The running buffer contained 0.24 M Tris base, 2.5 M glycine, and 0.1% SDS (pH 8.3). In addition, 1 mM thioglycolate and 1 mM sodium EDTA were added to the cathode (upper) buffer chamber. Sigma high molecular weight standards (29-116 kDa) were used to estimate molecular weights. Gels were stained with Coomassie Blue R or silver-stained using the procedure of Blum (16) , except that the concentrations of sodium thiosulfate and silver nitrate used were half of the published values.

Native Isoelectric Focusing PAGE

Purified isoprene synthase (Bio-Sil SEC 250-5 purified) was analyzed by isoelectric focusing (IEF) using 1.0-mm-thick precast Novex pH 3-7 IEF gels. The electrophoresis was performed at 4 °C to improve the resolution of the separation and to improve the recovery of active isoprene synthase from the gel. The 2 sample buffer contained 50 mM Tris-HCl (pH 8.0), 20 mM MgCl, 10% (v/v) PEG 400, and 2 mM DTT, and the electrode buffers used were Novex IEF anode buffer and Novex pH 3-7 IEF cathode buffer with 5 mM DTT (17). The gel was run as suggested by Novex (18) . Two lanes were saved for activity assays, and the remainder of the gel was fixed in 0.16 M sulfosalicylic acid, 11.5% (w/v) trichloroacetic acid for 30 min. The gel was then stained with Coomassie Blue R (0.1% (w/v) in 40% (v/v) methanol, and 10% (v/v) acetic acid) and destained with 25% (v/v) ethanol and 8% (v/v) acetic acid until the background was clear, about 20 min. The staining procedure was repeated one time, and the gel stored at room temperature in destain solution until used for two-dimensional electrophoresis. To locate isoprene synthase, the unstained portions of gel were cut into 2-mm slices, each placed in 4.8-ml vials with 10 µl of modified IPB (200 mM Tris-HCl (pH 8.0), 40 mM MgCl, 1% PEG 400, 5 mM DTT), and 5 µl of DMAPP solution as follows. Slices from lane 1 were incubated with 10 mM DMAPP for 20 min, and slices from lane 2 were incubated with 17 mM DMAPP for 10 min. Isoprene was measured by the GC/HgO method. The pH of each gel slice was estimated by comparing its distance from the cathode to IEF standards (Sigma and Pharmacia, pI 3-6.6) visible on the stained portion of the gel.

Two-dimensional Electrophoresis

Two-dimensional electrophoresis was performed as suggested by Novex (19) . A 1.0-mm-thick Novex 8-16% Tris-glycine two-dimensional well gel was used for the separation, using the same running and sample buffers as for SDS-PAGE. A 5.5-cm-long slice (pH 3.4 to 6.9) was loaded, and the gel was run at 125 V until the dye front had moved out of the IEF gel and into the stacking gel (10 min). The gel was then run for 2.25 h at room temperature and silver-stained as described above.

Preparation of Peptides with Cyanogen Bromide

Purified isoprene synthase (Bio-Sil SEC 250-purified) was separated by SDS-PAGE as described above. Protein was stained for 10 min with low-acid Coomassie (0.1% (w/v) Coomassie Blue R, 40% (v/v) methanol, and 1% (v/v) acetic acid) and destained for 20 min with 50% (v/v) methanol. The 62- and 58-kDa bands visible on the gel were excised with a razor blade and electroeluted with an electroelution cell designed and built in our laboratory, using the protocol recommended by Amicon for their Centrilutor Micro-Electroeluter (20) and the buffers used for SDS-PAGE. The eluted protein was desalted by diafiltration into water and concentrated to 100 µl. These samples were transferred to plastic microcentrifuge tubes and partially dried in a Savant Speed-Vac. One-hundred microliters of CNBr solution (30 mg/ml CNBr in 70% formic acid) were added to each sample, and the reaction mixtures were shaken gently in the dark at room temperature for 24 h. The cleavage reaction was stopped by the addition of 900 µl of water. To evaporate the formic acid, the water was removed by Speed-Vac, another 1 ml of water was added, and the sample was again brought to near dryness using the Speed-Vac. Fifty microliters of 1 Novex Tricine SDS sample buffer (with 100 mM DTT, 1 mM thioglycolate, and 1 mM sodium EDTA) were added to the samples in preparation for Tricine SDS-PAGE. Separation of the peptides, and their undigested parent polypeptides, was performed using a precast 16% Tricine gel (Novex) and Novex Tricine SDS running buffer with 1 mM thioglycolate and 1 mM sodium EDTA added to the cathode (upper) buffer chamber. The separated peptides were electroblotted at 40 V, 200 mA for 2 h at room temperature onto polyvinylidene difluoride membranes (Novex) using a Novex XCell blot module and blotting buffer containing 25 mM Bis-tris, 25 mM Bicine, 1 mM sodium EDTA, and 2 mM thioglycolate (pH 7.3). The membrane was rinsed with deionized water, soaked in methanol for a few seconds, stained with low-acid Coomassie for 1 min, destained with 50% (v/v) methanol for 10-15 min, rinsed extensively with deionized water, allowed to air-dry for several hours at room temperature, and stored at -20 °C in preparation for commercial amino acid sequencing.

NH-terminal Sequencing of Peptides

The 58- and 62-kDa bands and the CNBr cleavage peptides were NH-terminally sequenced commercially on a 473A protein sequencer (Applied Biosystems, Foster City, CA) using standard Edman chemistry (21) at the Macromolecular Resources Laboratory (Colorado State University, Fort Collins, CO). The amino acid sequences obtained from isoprene synthase were compared with other known protein amino acid sequences using the Blast data base search program at the National Center for Biotechnology Information (22) .

Metal Ion Dependence

Partially purified enzyme (Superdex 200 purified) was diafiltered into 50 mM Tris-HCl (pH 8.0), 1% (v/v) PEG 400, 2 mM DTT to remove magnesium ion. Fifteen-µl enzymatic reactions contained enzyme, 5 mg/ml bovine serum albumin, 10 mM DMAPP, and 0.1 mM to 30 mM MgCl, MnCl, ZnCl, or CaCl. Control reactions were identical except they contained no enzyme. Samples were incubated at 32 °C for 10 min and assayed for isoprene production by GC/HgO.


RESULTS

The goal of this work was to obtain highly purified isoprene synthase, and if possible, determine its partial amino acid sequence. Until this work, the enzyme had only been partially purified (6) , and several problems hampered further purification. First, the enzyme appears to be quite sensitive to denaturation due to dilution and oxidation, so it was necessary to keep the protein concentration high, and to include DTT in all buffers. Second, we found that glycerol stabilized the enzyme in early stages of purification, but that polyethylene glycol worked better at the latter stages of purification. Indeed, enzymatic activity was barely detectable after the hydroxyapatite column if glycerol was used in the column buffer instead of PEG 400. In addition, two chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase, copurified with isoprene synthase on the first two columns, complicating the purification of this minor protein.

Another complication in this work was the need to assay the enzyme and non-enzymatic control reactions by the appearance of the volatile product, isoprene. This was accomplished by headspace analysis of sealed reaction mixtures using rapid gas chromatography with detection of accumulated isoprene with a sensitive, reduction gas detector (23) . Under typical reaction conditions, the isoprene synthase headspace assay was linear with time for at least an hour, and the rates of the reaction were linear with respect to amount of enzyme added (data not shown). The non-enzymatic production of isoprene from DMAPP under the optimal assay conditions used (pH 8.0, 12 mM MgCl; Ref. 6) was insignificant. These results are important, because they show that linear initial rate data for the isoprene synthase reaction can be obtained with this assay, with little apparent inhibition of the reaction by buildup of the reaction product, isoprene, and that the non-enzymatic conversion of DMAPP to isoprene is negligible under these conditions.

Purification

summarizes the results from a typical purification of isoprene synthase from 150 g of aspen leaves. Isoprene synthase activity was too dilute to detect in the crude extract, so it was necessary to concentrate these preparations severalfold to get reproducible measurements of the activity. In addition, there was phosphatase activity that degraded the substrate. In subsequent purification steps, isoprene synthase activity was easily measured.

The enzyme was purified 2-fold by precipitation with ammonium sulfate at 40-55% saturation, with 95-100% recovery of activity. Isoprene synthase was retained on a Q-Sepharose column and eluted at 300 mM NaCl at the front of a large protein peak, yielding an approximately 3-fold purification. The enzyme was purified 25-fold by a Superdex 200 HR column, which separated isoprene synthase activity from a large protein peak (calculated molecular mass approximately 500 kDa), assumed to contain ribulose-bisphosphate carboxylase/oxygenase (Rubisco) (24) . The next column used, a hydroxyapatite column, was the critical step for purifying isoprene synthase. The enzyme eluted at approximately 170 mM phosphate and was purified approximately 10-fold. Until this step, the enzyme copurified with phosphoribulokinase and contained phosphatase activity that degraded DMAPP. The final step of routine purification was performed using an SEC-250 HPLC column. Fig. 1 shows that most of the protein loaded onto this column eluted coincident with isoprene synthase activity; the only other detectable proteins were a small shoulder peak at 8.5 min and a small peak at 12 min. Overall, a typical purification produced a 4000-fold increase in specific activity relative to crude enzyme and a 9% recovery of total enzymatic activity (). With other preparations, up to 6000-fold purifications were seen. Since only 50 µg of purified isoprene synthase were recovered from this large preparation (2250 mg of protein, ), isoprene synthase appears to be a minor cellular protein.


Figure 1: Purification of isoprene synthase by SEC-250 size exclusion chromatography. One-hundred µl of concentrated protein were loaded onto the column and eluted with IPB. Fifteen-µl samples incubated with 10 mM DMAPP for 10 min at 32 °C were assayed for isoprene production by GC/HgO. Isoprene synthase activity is expressed as peak area (microvolt seconds) from GC/HgO analysis.



In some experiments a Mono S cation exchange column was also employed. Purification data from these experiments are not shown in , because this column was not routinely used. While this column did separate isoprene synthase from contaminating proteins, enzymatic activity was partially lost on this column, and the specific activity did not increase significantly over the SEC-250 purification.

Identification of Isoprene Synthase by SDS-PAGE

Identification of the bands corresponding to isoprene synthase was determined by a process of elimination. Contaminant proteins in the purified enzyme preparations were ruled out by careful examination of electrophoresis gels across the peaks of activity from chromatographic columns (data not shown). A 62-kDa band clearly correlated with activity on all columns, and a 58-kDa band was present in the most active fractions. Fig. 2shows a silver-stained SDS-PAGE gel of isoprene synthase at various steps of purification. A constant amount of activity was loaded in each lane. The protein in lanes 5 and 6 had been enriched 4000-fold. As mentioned above, the Mono S column did eliminate some contaminant proteins; this can be seen in lane D. It is clear from this gel that the 58- and 62-kDa bands were highly enriched by the purification scheme and appear to be at least 90% pure. When stained with Coomassie Blue R, the most purified preparations appeared nearly homogeneous with only the 62- and 58-kDa bands visible (data not shown).


Figure 2: Analysis of isoprene synthase purification by SDS-PAGE. Equal amounts of isoprene synthase activity were loaded onto each lane of this silver-stained SDS-PAGE gel. Lane A, post Superdex-200; lane B, post-hydroxyapatite; lane C, post-SEC-250; lane D, post-Mono S. It can be seen in this gel that the 62- and 58-kDa bands (arrows) are greatly enriched by the purification scheme.



Native Mass and Subunit Composition

The native molecular mass of isoprene synthase was estimated from the elution volume of the enzyme on two different sizing columns. The apparent mass according to the Superdex 200 HR column was 98-107 kDa, based on Bio-Rad gel filtration standards. Sodium chloride was included with the column buffer to prevent peak tailing, but had no significant effect on the elution volume of isoprene synthase on this column. On the SEC-250 column, the elution volume of isoprene synthase activity varied significantly with salt concentration, from 7.4 ml with no added NaCl versus 7.76 ml with 300 mM NaCl. These elution volumes, when compared with standards, gave an estimated molecular mass of 217-137 kDa. As this behavior is expected with a negatively charged protein on a silica based column (25) , the estimate of 137 kDa is probably the most accurate from this column. Taken together, the data from these two sizing columns suggest that the native molecular mass of isoprene synthase is between 98 and 137 kDa. Since SDS-PAGE showed two bands correlating with isoprene synthase activity, at 58- and 62-kDa, isoprene synthase may be an heterodimer with a native molecular mass of approximately 120 kDa.

Isoelectric Focusing and Two-dimensional Electrophoresis

Native IEF gels were run on SEC-250-purified isoprene synthase. Portions of the gel were stained for protein, and the remainder of the gel was cut into slices and assayed for isoprene synthase activity. Fig. 3shows a Coomassie-stained IEF gel aligned with: recovered enzymatic activity from a sliced and assayed IEF gel (A) and a second dimension (two-dimensional) electrophoresis gel (B). Based on the recovered activity, isoprene synthase has a native pI of 4.7 (Fig. 3A). The stained gel shows a strong band with a pI of 4.7 and an apparent contaminant band with a pI of 4.9. A smearing of protein, correlating well with a smearing of enzyme activity, can be seen extending to pH 5.3. Because there were no solubilizing agents added to the gel or sample buffer, it is likely that this smearing of isoprene synthase was due to precipitation of the enzyme at this relatively low pH. Attempts to decrease the smearing with a non-ionic detergent, 1% (v/v) Nonidet P-40 (17, 18) , were not successful.


Figure 3: Alignment of IEF gel with recovered isoprene synthase activity and two-dimensional gel. A, SEC-250-purified enzyme was analyzed by IEF-PAGE. The recovered isoprene synthase activity from an unstained, sliced sample is aligned with a parallel, Coomassie-stained lane. Gel pH values were estimated from IEF standards run in a parallel lane. B, two-dimensional electrophoresis gel aligned with IEF gel above. The IEF gel was run in the second dimension on a 8-16% SDS-PAGE gel and silver-stained. Arrows indicate the 62- and 58-kDa bands.



Two-dimensional electrophoresis was used to verify the identification of isoprene synthase by SDS-PAGE (Fig. 3B). The two distinct spots (arrows) that correlate well with the observed activity on the first-dimensional gel are the 62- and 58-kDa bands described above. These data, when combined with the SDS-PAGE data discussed above, provide support for the 58- and 62-kDa bands being subunits of isoprene synthase.

Amino Acid Sequencing

Purified isoprene synthase was separated by SDS-PAGE, electroblotted onto polyvinylidene difluoride membranes, and NH-terminally sequenced commercially by Edman degradation. For this work, the 62-kDa subunit was designated ``'' and the 58-kDa subunit was designated ``.'' Amino terminus (NH terminus) sequencing of the subunit yielded 14 amino acid residues, but attempts at sequencing the subunit were unsuccessful. Like many eukaryotic proteins, it appeared to be chemically blocked. To unblock the NH terminus by removing acetylserine or acetylthreonine, the blotted subunit was exposed to trifluoroacetic acid vapors at 60 °C for 30 min. When this failed to unblock the protein, the blot was exposed to 0.6 M HCl at 25 °C for 24 h to remove formyl groups (26) . This treatment was also ineffective.

To obtain internal sequence data, the and subunits were isolated separately by SDS-PAGE and electroelution. The polypeptides were digested with cyanogen bromide (CNBr), and the resultant peptide fragments were NH-terminally sequenced commercially by Edman degradation. Cyanogen bromide cleavage was chosen over enzymatic cleavage, because it generally creates fewer, larger peptides than enzymatic methods, simplifying analysis. The subunit yielded primary peptides of 29, 13, and 6 kDa (1, 2, and 3), and the subunit yielded primary peptides of 25, 13, and 6 kDa (1, 2, and 3). The NH-terminal sequence of each peptide is shown in . Like the subunit, 1 yielded insufficient data to derive a sequence and appeared to be chemically blocked. 1 had an NH-terminal sequence very similar to the NH terminus of the subunit, confirming that this peptide is the NH terminus of the parent 58-kDa polypeptide. The discrepancy at residue 6 (L versus N) was probably due to a very low signal from 1 and does not indicate that they are necessarily different. The NH terminus of 2 was identical to the NH terminus of 2, and the NH terminus of 3 was identical to the NH terminus of 3, suggesting that and are closely related; it is not known at this point if the two subunits are coded for by the same gene or if they are coded for by two very similar genes.

Isoprene synthase sequences were compared with other known protein sequences using the Blast data base search program (22) . No identical matches were made to any of the three peptide sequences entered. The NH terminus of the 58-kDa subunit did share a 36% identity with an internal sequence of the 70-kDa precursor to limonene synthase, which uses geranyl diphosphate, a C analog of DMAPP, as its substrate (27) . However, neither of the other peptide sequences shared any homology with limonene synthase, and it appears unlikely these two proteins are closely related. In addition, the partial sequences obtained from isoprene synthase do not share homology with other known prenyl diphosphate-utilizing enzymes, and it therefore appears that isoprene synthase is not closely related to these other well studied enzymes (28, 29, 30) .

Biochemical Properties

Like other enzymes that use allylic diphosphates as substrates (31, 32, 33, 34, 35, 36) , the isoprene synthase reaction requires a divalent cation for activity. When divalent cations were removed by diafiltration into metal-free buffer, no enzymatic activity was observed. Upon the addition of magnesium or manganese ion, enzymatic activity increased with metal concentration with maximal activity at 5 mM Mn or 10 mM Mg (data not shown). Higher levels of manganese were inhibitory to the enzymatic reaction; it may be that manganese binds both the substrate and the active site, preventing substrate binding to the enzyme (35) . In contrast with Mg and Mn, Zn and Ca were ineffective enzymatic cofactors. This behavior is typical of other enzymes that bind allylic diphosphate substrates (31, 36) .

As mentioned above, the enzyme was quite sensitive to oxidation, and high levels of DTT were required for the successful extraction and purification of active enzyme. This suggests that reduced sulfhydryl groups are important for enzyme structure or catalysis. To explore this further, purified isoprene synthase was exposed to two oxidizing reagents, sodium tetrathionate and oxidized glutathione, and to two reagents that covalently modify cysteine groups, iodoacetamide and N-ethylmaleimide. All four reagents inhibited the isoprene synthase reaction at 1-3 mM, although the observed inhibition ranged from 10% by 1 mM oxidized glutathione to nearly 90% by 3 mMN-ethylmaleimide. These data are consistent with the hypothesis that free thiols are necessary for enzymatic activity. The need for free thiols has also been observed in other enzymes that use prenyl diphosphate substrates (31, 33, 37) .

Stoichiometry

The stoichiometry of the enzymatic reaction was investigated by measuring the ratio of substrate used to isoprene and pyrophosphate made. In addition, the enzymatic reaction was assayed for allylic alcohol formation. Partially purified preparations of enzyme produced significant levels of allylic alcohols. However, highly purified enzyme produced no detectable alcohols and produced isoprene and pyrophosphate in similar amounts. The average ratio of isoprene produced to DMAPP consumed was 1.25 ± 0.3. The average ratio of isoprene to pyrophosphate produced was 1.0 ± 0.2. These data suggest that the enzyme stoichiometrically converts DMAPP to isoprene plus pyrophosphate.

Kinetic Properties

At pH 8, the pH optimum of the enzyme (6) , the enzymatic reaction reached maximum velocity (900 nmol isoprene min mg protein) at 10 mM DMAPP, but was inhibited by higher levels of substrate. Thus, it was difficult to measure the true V and K for the reaction. The apparent K for DMAPP was quite high at 8 mM, whereas the turnover number, k = 1.7 s, was low but similar to that of other enzymes that use prenyl-diphosphate substrates (31, 38, 39) . Because the K is very high, and the k is low, the k/K ratio for isoprene synthase was 215, which is at least 2 orders of magnitude lower than other enzymes that use similar substrates, indicating that this preparation of isoprene synthase is a very inefficient enzyme in vitro.


DISCUSSION

The published theories of isoprene formation in animals and animals suggest that the production of this hydrocarbon is non-enzymatic. In the 1980s, Deneris et al.(40) investigated the formation of isoprene by rat liver extracts. Isoprene was produced upon the addition of mevalonate and ATP to liver extracts, presumably from IPP or DMAPP that was enzymatically produced from the added mevalonate. Upon acidification of these same extracts, isoprene production increased at least 60-fold. They also performed studies with the alcohol analogs of IPP and DMAPP, 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol, and found that acidification of the 3-methyl-2-buten-1-ol (DMAPP analog) yielded significant isoprene, whereas acidification of 3-methyl-3-buten-1-ol (IPP analog) did not. Thus, they concluded that DMAPP is the predominant mevalonate-derived product that undergoes an acid-catalyzed elimination of pyrophosphate, yielding a transient carbocation that would give rise to isoprene as well as solvolysis products, 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol. These reactions are shown in Fig. 4. Because conversion of the carbocation to isoprene is essentially irreversible, the authors concluded that some isoprene would be produced in vivo, even at neutral pH.


Figure 4: Scheme for isoprene formation from DMAPP by isoprene synthase or acid catalysis. The isoprene synthase reaction is shown on the top of the scheme. An acid-catalyzed reaction is shown to proceed through a delocalized carbocation that may eliminate a proton to produce isoprene or be hydrated to form a primary alcohol (3-methyl-2-buten-1-ol) or more stable tertiary alcohol (2-methyl-3-buten-2-ol). As discussed in the text, neither alcohol is produced by purified isoprene synthase, but the carbocation shown may be an enzymatic intermediate.



Sanadze has suggested that acid-catalyzed rearrangement of DMAPP is the origin of isoprene in plants as well (3) . In the light, the chloroplast thylakoid lumen is acidified to approximately pH 5 by proton pumping (7) , and if DMAPP is contained in this compartment, the acid-catalyzed reaction described above could generate isoprene. As shown in Fig. 4, such reactions would also generate 3-methyl-2-buten-1-ol and 2-methyl-3-buten-2-ol; we have been unable to detect these alcohols in illuminated aspen leaves that are producing isoprene.() There is also no published evidence that DMAPP is located in the thylakoid lumen. As discussed by Sharkey et al.(2) , Sanadze's explanation leads to the conclusion that isoprene formation is merely a metabolic leak, but does not explain why some plants emit large amounts of isoprene and others emit essentially none.

We decided to look directly for an enzyme in leaves that catalyzed the formation of isoprene from DMAPP and after several unsuccessful attempts succeeded in extracting such an enzyme, which we termed isoprene synthase (6) . An assay system, based on gas chromatography with a mercuric oxide reducing gas detector (23) , was also an important technical development that allowed routine quantitation of the activity of this enzyme. In the work reported here, a purification scheme was developed that yielded 4000-6000-fold purification of the enzyme and up to 8% final recovery of activity. The only observed reaction products of the purified enzyme with DMAPP were isoprene and inorganic pyrophosphate. The ratios of DMAPP consumed to isoprene and pyrophosphate produced were nearly 1:1, confirming that DMAPP is converted stoichiometrically to isoprene and pyrophosphate. The purified enzyme produced no detectable amounts of the C alcohols shown in Fig. 4, indicating that it is unlikely that the enzymatic reaction proceeds via an intermediate alcohol.

The catalytic mechanism of isoprene synthase is not yet known, but may be analogous to terpene cyclases. For example, limonene synthase from spearmint (27) and pinene cyclases from sage (41) convert geranyl diphosphate to cyclic monoterpenes, but also produce minor acyclic products, including the hydrocarbons myrcene and ocimene. These minor products are C-hydrocarbon analogs of isoprene, and their formation could be a paradigm for the mechanism of isoprene synthase. As proposed by Croteau and co-workers (34, 41) , myrcene and ocimene arise by proton elimination from transient carbocations formed by the ionization of geranyl diphosphate. Isoprene might arise similarly from the C allylic carbocation shown in Fig. 4. The role of Mg (or Mn) ion in the isoprene synthase reaction might be like that proposed for pinene cyclase: to neutralize the negative charge of the diphosphate moiety and thereby assist in ionization of the allylic substrate (34) . Future work may reveal whether the catalytic mechanism of isoprene synthase parallels that of terpene cyclases, or of DMAPP-prenyltransferases, which are also thought to convert DMAPP to an intermediate allylic carbocation (42) .

The purified isoprene synthase was found to share many properties with known terpene cyclases. As reviewed by Alonso and Croteau (36) , most of these latter enzymes require divalent cation (Mg or Mn) for activity, have pI values between 4 and 5, have pH optima between 6 and 8, are sensitive to thiol reagents, and have turnover numbers in the range of 0.01-1.0 s. Isoprene synthase requires Mg or Mn for activity, has a pI of 4.7, has a pH optimum near 8, is sensitive to thiol reagents, and has a turnover number of 1.7 s. In addition, many of these enzymes are dimeric, with subunits of 37-50 kDa. Isoprene synthase also appears to be a dimer, but unlike these other proteins which are homodimers, it appears to be a heterodimer with 62- and 58-kDa subunits. In spite of these similarities, the partial amino acid sequences obtained for isoprene synthase showed little or no homology to known prenyl diphosphate-utilizing enzymes; however, only a relatively small amount of isoprene synthase sequence information was available for comparison.

Purified isoprene synthase exhibited a relatively high K (8 mM) for its substrate, DMAPP. This result is quite puzzling. The substrate K values for known prenyl diphosphate-utilizing enzymes are in the µM range (31, 32, 33, 34, 35, 36) . Although the levels of DMAPP in plant cells have not been reported, it seems unlikely that they could be in the range of 8 mM. Some possible explanations are (a) that a cofactor was removed during isoprene synthase purification, (b) a low affinity form of the enzyme without an activating covalent modification was isolated, (c) the extraction and purification procedures reduced enzymatic activity through proteolysis or loss of quaternary structure, or (d) that isoprene synthase may be membrane bound and released during isolation as a low affinity form. With respect to d, it is noteworthy that some prenyl diphosphate-utilizing enzymes are thought to be associated with the endoplasmic reticulum and are solubilized by extraction procedures similar to the one used here for isoprene synthase (36) . Another possibility is that isoprene synthase may actually be a prenyltransferase that reacts DMAPP with an unknown cosubstrate, X. In the absence of X, the enzyme may produce isoprene instead of the normal prenylated product. These possibilities are under current investigation.

Despite the low catalytic efficiency of the purified isoprene synthase, there are several lines of evidence to suggest this enzyme is responsible for the majority of isoprene production in plants. First, when isoprene emissions from velvet bean leaves of different ages were compared with extracted isoprene synthase activity from these leaves, a clear correlation was seen (5) . Young leaves, which produced little isoprene, had very low isoprene synthase activity, whereas older isoprene-producing leaves had corresponding higher levels of isoprene synthase activity. With the exception of the oldest leaves, the amount of isoprene produced by leaves correlated very well with the amount of extractable enzyme activity, showing that levels of active isoprene synthase can account for observed isoprene emission rates. Second, the temperature optima for isoprene synthase and leaf isoprene emission are similar, indicating the two activities are related (10) . If isoprene production was due to a non-enzymatic conversion of DMAPP to isoprene, and the catalysis rate was controlled only by photosynthetically driven changes in DMAPP concentration or thylakoid pH, then emission rates would parallel photosynthesis at different temperatures. As emission rates do not parallel photosynthesis, and do parallel isoprene synthase activity, it appears that this enzyme ultimately controls observed emission rates. Finally, as mentioned in the Introduction, we have detected isoprene synthase activity in leaf extracts of several other isoprene emitting plants, showing that the enzyme is not limited to aspen leaves. In contrast, we have been unsuccessful at extracting isoprene synthase activity from non-emitting species of plants, indicating these plants may not contain the enzyme.

There are still many unanswered questions about isoprene synthase and isoprene formation in plants, including the regulation of isoprene synthase gene expression during leaf development, the subcellular location of the enzyme, and the mechanism of light activation of isoprene synthesis. As information is obtained on these points, we may gain a better understanding of a more general question: what is the physiological role of isoprene synthase in plants?

  
Table: Purification of aspen leaf isoprene synthase

Based on 150 g fresh leaves.


  
Table: Partial amino acid sequences of isoprene synthase subunits and isolated CNBr peptides

The 62- and 58-kDa subunits were designated and , respectively.



FOOTNOTES

*
This work was supported National Science Foundation Grants ATM-9007849 and ATM-9312153 and United States Environmental Protection Agency Grant R-815995-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Paravax Biopharmaceuticals, 1825 Sharp Point Dr., Fort Collins, CO 80525.

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215. Tel.: 303-492-7914; Fax: 303-492-5894.

The abbreviations used are: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; HPLC, high performance liquid chromatography; GC/HgO, gas chromatography/mercuric oxide detection; GC/FID, gas chromatography/flame ionization detection; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; IPB, isoprene synthase purification buffer; PEG-400, polyethylene glycol 400; DTT, dithiothreitol; PEB, plant extract buffer; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Bicine, N,N-bis(2-hydroxyethyl)glycine.

M. Nemecek-Marshall, B. Taylor, M. Wildermuth, and R. Fall, unpublished observations.

G. Silver and R. Fall, unpublished observations.


ACKNOWLEDGEMENTS

We thank Cindy Bozic, Rob Kuchta, Jennifer Kuzma, Craig Miles, Michele Nemecek-Marshall, Dale Poulter, and Mary Wildermuth for their valuable advice. We also thank Cheryl Wojciechowski for help with the preparation of leaf extracts.


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