From the
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
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
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,
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
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) .
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
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.
To obtain internal sequence data,
the
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
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) .
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.
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
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
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
Purified isoprene synthase exhibited a
relatively high K
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?
Based on 150 g fresh leaves.
The 62- and 58-kDa
subunits were designated
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
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.).
(
)
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.
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.
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.
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
The
58- and 62-kDa bands and the CNBr cleavage peptides were
NH-terminal Sequencing of Peptides
-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.
; 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.
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.
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.
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) .
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.
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.
alcohols shown in Fig. 4,
indicating that it is unlikely that the enzymatic reaction proceeds via
an intermediate alcohol.
-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) .
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.
(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.
Table:
Purification of aspen leaf isoprene
synthase
Table:
Partial amino acid sequences of isoprene
synthase subunits and isolated CNBr peptides
and
, respectively.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.