From the
A novel
S-adenosyl-
L-methionine:halide/bisulfide
methyltransferase (EC 2.1.1.-) was purified approximately
1000-fold to apparent homogeneity from leaves of Brassica
oleracea. The enzyme catalyzed the
S-adenosyl-
L-methionine-dependent methylation of the
halides iodide, bromide, and chloride to monohalomethanes and of
bisulfide to methanethiol. The dual function of the enzyme was
demonstrated through co-purification of the halide- and
bisulfide-methylating activities in the same ratio and by studies of
competition between the alternative substrates iodide and bisulfide.
The purification procedure included gel filtration, anion exchange
chromatography, and affinity chromatography on adenosine-agarose.
Elution of the protein from a chromatofocusing column indicated a pI
value of 4.8. The pH optimum of halide methylation (5.5-7.0) was
different from that of bisulfide methylation (7.0-8.0). The
molecular mass values for the native and denatured protein were 29.5
and 28 kDa, respectively, suggesting that the active enzyme is a
monomer. The enzyme had the highest specificity constant for iodide and
the next highest for bisulfide. Substrate interaction kinetics and
product inhibition patterns were consistent with an Ordered Bi Bi
mechanism.
Interest in trace gas emissions has increased over the last two
decades following the realization of their impact on atmospheric
chemistry
(1) . Halogen- and sulfur-containing organic gases
have attracted particular attention because of their respective effects
on the integrity of stratospheric ozone
(2) and the formation
of acid rain
(3) .
Biological activity in the oceans is
commonly viewed as a major source of organohalogen
(4, 5) and organosulfur emissions
(6, 7) .
Terrestrial microorganisms, fungi, and a few higher plants also emit
these gases
(8, 9) . The biochemical bases for these
emissions, however, remain poorly understood. Possible mechanisms
involve spontaneous
(10) or enzymatic
(11, 12) reactions. A haloperoxidase-mediated incorporation of
halides into a carbon skeleton was suggested as the main route for
halomethane biosynthesis
(11) but has never been actually shown
to produce monohalomethanes. Subsequently, an enzymic mechanism
involving a single-step conversion of halides into a monohalocarbon was
proposed
(13) ; this was supported by evidence for incorporation
of the methionine methyl group into a monohalomethane
(14) .
Recently, it was reported
(15) that a methyl chloride
transferase from the marine red alga Endocladia muricata could
carry out the transfer of a methyl group from
S-adenosyl-
L-methionine (AdoMet)
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Preliminary evidence was also presented for a similar activity
in a wood-rotting fungus, Phellinus pomaceus, and a halophytic
higher plant, Mesembryanthemum crystallinum.
Methanethiol
was originally thought to come from methionine via a methioninase
(
L-methionine methanethiol-lyase) reaction
(12) .
However, the observation that several bacterial isolates from soil and
agricultural crops as well as cultures of marine algae evolved
CH
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
A survey of higher plants done in our laboratory
(20) showed that many diverse species produce CH
Plant MaterialPlants of Brassica oleracea cv. April Red were grown in a
greenhouse under a photoperiod of at least 12 h and were watered and
fertilized as necessary. The two most expanded basal leaves of
3-month-old plants were used for enzyme extraction. Chemicals S-Adenosyl-
L-methionine was from Boehringer Mannheim.
The inorganic phosphorus test kit,
S-adenosyl-
L-homocysteine, 5`-ADP-agarose, KI, KBr,
and KCl were purchased from Sigma. Ammonium sulfide and authentic
CH
Alternative substrate competition experiments were
performed by varying the concentration of one substrate at each of a
series of concentrations of the other. The concentration of AdoMet was
kept constant at 0.5 m
M. Data were presented as
double-reciprocal plots of initial velocity (v) versus varying substrate (S) concentrations.
Substrate interaction
studies were done by fixing the concentration of one substrate while
changing that of the other. Linear regressions were fitted to the data
in double-reciprocal plots. Replots of the data were used to determine
the kinetic parameters.
B. oleracea leaf extracts catalyzed the
AdoMet-dependent methyl transfer to X
The halide and bisulfide methyltransferase activities from
B. oleracea were purified to apparent homogeneity. The two
activities co-purified at a constant ratio (1.26 ± 0.14)
throughout the procedure (, Figs. 2 and 3). The kinetics of
mutual inhibition between the substrates I
The halide/bisulfide methyltransferase
exhibited different pH optimum values for its alternative halide
(Fig. 5 A) and bisulfide (Fig. 5 B)
substrates. A likely reason for this difference is the pH dependence of
the HS
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
to a halide (X
) as follows.
SH in the presence of bisulfide (HS
)
suggested that this gas may also be produced via a different mechanism
(16) . Thiol methyltransferases, capable of catalyzing the
following reaction, were later found in a variety of organisms
including bacteria
(16) , algae
(17) , and mammals
(18, 19) .
I
when supplied with I
, and CH
SH when
supplied with HS
, in a manner similar to Reactions 1
and 2. The levels of the two activities were highly correlated among
species. Plants from the family Brassicaceae exhibited some of the
highest rates of CH
I and CH
SH production. To
elucidate the biochemical basis for these environmentally important gas
emissions, we developed an easy and reliable assay for their
quantitation and proceeded to purify and characterize the halide- and
bisulfide-methylating activities.
SH, CH
Br, and CH
I were obtained
from Aldrich. Authentic CH
Cl was purchased from Liquid
Carbonic (Scarborough, Ontario, Canada). Sephadex G-100, Sephadex G-25,
gel filtration calibration kit, and Polybuffer 74 were from Pharmacia
Biotech Inc. Calf intestinal alkaline phosphatase was purchased from
New England Biolabs (Beverly, MA). The protein dye reagent, TEMED,
ammonium persulfate, and bisacrylamide were all from Bio-Rad
(Mississauga, Ontario, Canada). All other chemicals were of reagent
grade. BuffersThe buffers used were as follows: 100 m
M Tris acetate, pH 7.5,
containing 10% glycerol (v/v) and 14 m
M 2-mercaptoethanol
(buffer A); 25 m
M Tris acetate, pH 7.4, containing 10%
glycerol and 14 m
M 2-mercaptoethanol (buffer B); 25
m
M Tris acetate, pH 7.4, containing 14 m
M
2-mercaptoethanol (buffer C); buffer C with 100 m
M NaCl
(buffer D); 25 m
M BisTris/iminodiacetic acid, pH 7.0 (buffer
E); and Polybuffer 74/ iminodiacetic acid (1:10, v/v), pH 4.0 (buffer
F). All buffers were filtered before use through a 0.22-µm
membrane. Measurement of Halide/Bisulfide Methyltransferase Activity
Enzyme Assay
Since Iwas the preferred
substrate among halides, it was used as the halide substrate throughout
the purification procedure. The enzyme activity was assayed in a 1-ml
mixture containing 0.5 m
M AdoMet, 50 m
M KI (halide
methylation), or 20 m
M (NH
)
S
(bisulfide methylation) all prepared in buffer A. Enzyme preparations
containing up to 200 µg of protein were used to start the reaction.
The mixture was incubated in a 5-ml glass vial sealed with a screw cap
fitted with a Teflon-lined septum (Supelco, Oakville, Ontario, Canada)
and maintained on an orbital shaker (150 rpm) at room temperature. The
reaction rate was linear for at least 45 min at all enzyme and
substrate concentrations. A standard incubation time of 30 min was
adopted.
Gas Chromatography
The products formed were
analyzed by gas chromatography using a flame ionization detector.
One-ml headspace samples were injected in a 210 0.3-cm
stainless steel column packed with 80/100-mesh Porapak Q (Supelco) in a
Hewlett-Packard 5890 series II gas chromatograph. Column temperatures
were 160 °C for CH
I and CH
Br, 145 °C
for CH
SH, and 130 °C for CH
Cl. The carrier
gas (helium, ultrapure) flow rate was 40 ml/min. The column was purged
by heating to 200 °C between injections. Products were quantified
by peak area and identified by comparison of their retention times with
those of authentic methyl halides or CH
SH, which were used
to calibrate the instrument. Recoveries of 75 nl of CH
I and
28 nl of CH
SH injected to vials containing the assay
mixture were 69 and 87%, respectively, after 30 min. The data presented
here were not corrected for recoveries. The identity of CH
I
and CH
SH in the headspace of enzyme assays was confirmed by
mass spectrometry using a KRATOS MS 50 mass spectrometer operated in
the electron impact mode. Protein EstimationTotal soluble proteins were determined by the method of Bradford
(21) using the Bio-Rad protein reagent, following the microassay
procedure, and using bovine serum albumin as the standard. Preparation of Adenosine-Agarose Affinity Gel5`-ADP-agarose (4 ml) was washed under vacuum according to the
manufacturer's instructions and incubated with 800 units of calf
intestinal alkaline phosphatase and 5 ml of calf intestinal alkaline
phosphatase buffer (1
), pH 7.5, in a total volume of 9 ml. The
gel was dephosphorylated overnight at 37 °C in a continuously
rotating reaction vial, transferred to a column, washed with 10 ml of
buffer C containing 2
M NaCl, and washed again with 20 ml of
buffer C alone. The washing was done in fractions of
10 ml, which
were subsequently assayed for phosphate content. The dephosphorylation
reaction was repeated until no more phosphate was detected. The gel was
washed with an excess of deionized water before each reaction. Free
phosphate was determined colorimetrically with the method of Fiske and
Subbarow
(22) . Purification of Halide/Bisulfide Methyltransferase
Extraction
All manipulations were carried out at 4
°C unless stated otherwise. Halide- and
HS-methylating activities were monitored
simultaneously throughout the purification procedure. Generally, 1 kg
of freshly harvested leaves were cut into small squares, immediately
frozen with liquid nitrogen, and then homogenized with buffer A (1:3,
w/v) and polyvinylpolypyrrolidone (10%, w/w) for 5 min in a blender at
full speed. The homogenate was filtered through eight layers of
cheesecloth and centrifuged for 20 min at 15,000
g.
The supernatant (crude extract) was recovered, and enzyme activity and
protein concentration were assayed in an aliquot desalted by passage
through a PD-10 column (Pharmacia). The crude extract was fractionated
with solid (NH
)
SO
, and the proteins
that precipitated between 60 and 85% saturation, containing halide and
bisulfide methyltransferase activities, were recovered by
centrifugation at 12,000
g for 20 min.
Gel Filtration on Sephadex G-100
The pellet from
the 60-85% (NH)
SO
fraction
was resuspended in 15 ml of buffer B and applied to a Sephadex G-100
column (2.6
90 cm) that had been previously equilibrated in the
same buffer. The column was eluted with buffer B at 25 ml/h, and 2-ml
fractions were collected. The active fractions from this step were
pooled and concentrated by diafiltration in a stirred Amicon cell
fitted with a YM-30 membrane (Amicon, Danvers, MA).
Affinity Chromatography on Adenosine-Agarose
The
concentrated sample was applied to an adenosine-agarose column (1.5
3 cm) that had been previously equilibrated with buffer B. The
column was washed at a constant flow rate of 10 ml/h with 15 ml of
buffer B, followed by 8 ml of the same buffer containing 300
m
M (NH
)
SO
. The salt was
then removed by washing the column with buffer B. The halide- and
bisulfide-methylating activities were recovered by elution with a 40-ml
linear gradient of 0-4 m
M AdoMet in buffer B. One-ml
fractions were collected and assayed for activity and protein content.
The halide and bisulfide methyltransferase activities eluted between 2
and 3 m
M AdoMet. The remaining bound proteins were washed off
the column with 2
M NaCl in buffer B; no methyltransferase
activity was detected in this fraction.
Anion Exchange on Protein Pak Q
Active eluate from
the affinity step was submitted to high performance liquid
chromatography (HPLC) (Millipore, Milford, MA) on a 1 10-cm
Protein Pak Q anion exchange column (Millipore) equilibrated with
buffer C. The sample was loaded at a flow rate of 0.5 ml/min, and the
column was washed with the same buffer until no further UV-absorbing
material was eluted. Enzyme activity was then eluted with NaCl in
buffer C in a two-step linear gradient of 0-200 m
M in 80
min and 200-500 m
M in 20 min, collecting 0.5-ml
fractions.
Gel Filtration on Superdex 75
The fraction
containing the first peak of halide and bisulfide methyltransferase
activity from the previous step was loaded on an HPLC Superdex 75 gel
filtration column (1 30 cm) (Pharmacia) previously equilibrated
with buffer D. Proteins were eluted in the same buffer at a flow rate
of 0.5 ml/min, collecting 0.5-ml fractions. At this level of
purification the halide and bisulfide methyltransferase preparation was
apparently free from contaminating proteins. ChromatofocusingAn enzyme preparation from the Sephadex G-100 gel filtration step was
subjected to HPLC chromatofocusing on a Mono P HR (5/20) column
(Pharmacia) previously equilibrated with buffer E. Unbound proteins
were removed by washing the column with the same buffer. Enzyme
activity was then eluted with a pH gradient of 7.0-4.0 generated
with 45 ml of buffer F at a flow rate of 1 ml/min. One-ml fractions
were collected in 0.2 ml of 500 m
M Tris acetate buffer, pH
7.7, containing 50% glycerol. Molecular Weight DeterminationMolecular weight of the native halide/bisulfide methyltransferase was
determined by gel filtration on a Sephadex G-100 column (2.5
44
cm) calibrated with the following markers: ribonuclease A
( M
13,700), chymotrypsinogen A ( M
25,000), ovalbumin ( M
43,000), and albumin
( M
67,000). The column void volume was determined
with blue dextran 2000. Buffer B was used for column equilibration and
elution. One-ml samples were applied to the column, which was operated
at a flow rate of 20 ml/h. In order to achieve better resolution,
column calibration was done in two steps, the first involving
ribonuclease A and ovalbumin and the second involving albumin and
chymotrypsinogen A. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Proteins from different purification steps were separated by SDS-PAGE
under denaturing conditions
(23) using 12% acrylamide gels.
Gels were stained with silver nitrate or Coomassie Blue. The molecular
mass of the denatured enzyme was determined from a plot of log
molecular mass against the migration distance of standard proteins. Determination of Kinetic PropertiesThe kinetic properties of the halide/bisulfide methyltransferase were
determined using a preparation purified through a Sephadex G-100 gel
filtration step.
or
HS
. The enzyme responsible for both of the reactions
was purified to homogeneity, and its dual function was demonstrated.
Purification of Halide/Bisulfide Methyltransferase
The
enzyme was extracted from B. oleracea by subjecting leaf
tissue to a freeze-thaw cycle prior to homogenization and was purified
using (NH)
SO
precipitation followed
by gel filtration chromatography on Sephadex G-100, affinity
chromatography on adenosine-agarose, and HPLC on Protein Pak Q anion
exchange and Superdex 75 gel filtration columns. The specific activity
of the enzyme was enriched more than 1000-fold over the crude
preparation with a recovery of 0.35%. summarizes the
purification procedure, and Fig. 1shows a typical pattern of
proteins from various purification steps separated on SDS-PAGE and
visualized by Coomassie Blue.
Figure 1:
SDS-PAGE of fractions with
halide/bisulfide methyltransferase activity from the successive steps
of purification. Lane A, crude extract (10 µg);
lane B, 60-85% (NH
)
SO
(
5 µg); lane C, Sephadex G-100 (
5 µg);
lane D, Amicon YM-30 (
5 µg); lane E,
adenosine-agarose (
5 µg); lane F, Protein Pak Q
(
5 µg); lane G, Superdex 75 (
1 µg). The
molecular mass markers are indicated to the left in kDa. The
gel was stained with high sensitivity Coomassie Brilliant Blue
G-250.
Affinity chromatography on
adenosine-agarose was efficient in removing most of the contaminating
proteins (Fig. 2). Further purification was achieved by anion
exchange HPLC, which resolved four peaks of halide/bisulfide
methyltransferase activity (Fig. 3). The first and largest peak of
activity eluting from this column contained only two proteins as seen
on the SDS gel (Fig. 1). Further purification of this peak by HPLC on a
Superdex 75 gel filtration column resolved two protein peaks, only one
of which contained halide/bisulfide-methylating activity. This peak
appeared free of contaminants when visualized on SDS-PAGE
(Fig. 1). The Superdex-purified halide/bisulfide
methyltransferase migrated as a single band on SDS-PAGE with an
apparent molecular mass of 28,000 daltons. Since this is close to the
value of 29,500 determined for the native protein on a G-100 gel
filtration column, the active enzyme probably exists as a monomer.
Figure 2:
Elution of halide/bisulfide
methyltransferase from an adenosine-agarose affinity column.
Specifically bound proteins were eluted in a 0-4 m
M
AdoMet ( SAM) gradient in buffer B. Fractions of the eluate
were assayed for halide () and bisulfide (
)
methyltransferase activities. Both activities overlapped up to fraction
55. Proteins were continuously monitored by absorbance at 280 nm
( solid line). Most of the increase in the absorbance with the
gradient elution was due to the presence of AdoMet. nkat,
nanokatals.
Demonstration of Dual Activity
The halide and
bisulfide methyltransferase activities co-purified to homogeneity in
the same ratio (1.26 ± 0.14) during a five-step procedure
(, Figs. 2 and 3). Moreover, double-reciprocal plots of the
data from competition kinetics between the halide and bisulfide
substrates gave regression lines that intersected at the 1/ v axis (Fig. 4, A and B). These results
established that the halide and bisulfide methylations are catalyzed by
the same protein, halide/bisulfide methyltransferase.
Effect of pH on Halide/Bisulfide
Methyltransferase
The halide/bisulfide methyltransferase
activity was assayed over a wide range of pH, using buffers with
overlapping ranges. The pH optimum for Imethylation
was fairly broad, between 5.5 and 7.0 (Fig. 5 A), whereas that
for HS
methylation was sharper and more alkaline,
between 7.0 and 8.0 (Fig. 5 B). In addition, halide/bisulfide
methyltransferase activity eluted at pH 4.8 upon chromatofocusing on a
Mono P column, suggesting that the enzyme has a pI value of 4.8
± 0.2.
Enzyme Stability
After Sephadex G-100 gel
filtration, the enzyme was stable for over 2 months at -80 °C
in buffer B. After affinity chromatography, the halide/bisulfide
methyltransferase became extremely labile, losing all activity after
overnight storage at -80 °C. Addition of 20% glycerol to the
preparation and storage at -20 °C conserved 12% of the
activity after 48 h. In contrast, after the Protein Pak Q anion
exchange step, the enzyme retained more than 70% of its activity after
24 h and 55% after 48 h at 4 °C in buffer C containing 175
m
M NaCl. Kinetic Analysis
Substrate Interaction Kinetics
Double-reciprocal
plots with Ias the variable substrate at several
fixed concentrations of AdoMet gave converging lines (Fig.
6 A). The same pattern was observed when HS
was the variable substrate at different fixed concentrations of
AdoMet (Fig. 6 B).
Figure 6:
Double-reciprocal plots of initial
velocity ( v) versus substrate concentration for
the methylation of iodide and bisulfide. The effect of iodide
( A) or bisulfide ( B) concentrations on the initial
velocity of the methylation reaction at different fixed concentrations
of AdoMet ( SAM) is shown. nkat,
nanokatals.
Product Inhibition Kinetics
The order of substrate
binding and product release was determined from product inhibition
studies. S-Adenosyl-
L-homocysteine was a competitive
inhibitor with respect to AdoMet (Fig. 7 A) and noncompetitive
with respect to iodide (Fig. 7 B) or bisulfide
(Fig. 7 C). The kinetics of product inhibition for the
gaseous CHI or CH
SH could not be determined
under the present assay conditions.
Figure 7:
Product inhibition of halide/bisulfide
methyltransferase reaction, presented as double-reciprocal plots.
A, inhibition of the methylation reaction by
S-adenosyl-
L-homocysteine ( SAH) with respect
to S-adenosyl-
L-methionine; the concentration of
iodide was fixed at 50 m
M. B, inhibition of the
methylation reaction by S-adenosyl-
L-homocysteine
with respect to iodide; the concentration of AdoMet was fixed at 400
µ
M. C, inhibition of the methylation reaction by
S-adenosyl-
L-homocysteine with respect to bisulfide;
the concentration of AdoMet was fixed at 400 µ
M.
nkat, nanokatals.
As calculated from replots of
the data, the Vfor the formation of
CH
I was 1140 nanokatals/mg of protein; that for
CH
SH was 804 nanokatals/mg of protein. The respective
K
values for I
and
HS
were 1.3 and 4.7 m
M. There were two
different K
values for AdoMet depending
on the second substrate used, 0.03 m
M with I
and 0.226 m
M with HS
. Table II
summarizes the kinetic parameters of the halide/bisulfide
methyltransferase for its different substrates.
and
HS
were characteristic of competitive inhibition
(Fig. 4, A and B). Taken together, these
results establish that both halide- and bisulfide-methylating
activities reside on the same active site of a single protein, the
halide/bisulfide methyltransferase. The homogeneous protein was
obtained through a five-step procedure that resulted in an overall
purification of approximately 1000-fold with a recovery of 0.35%
(). Affinity chromatography on an adenosine-agarose column
was the key step, in which the enzyme specifically bound to the matrix
while most of the contaminating proteins were washed through during
sample loading (Fig. 2). The enzyme was selectively eluted with the
co-substrate AdoMet in a linear gradient resulting in a 1300-fold
increase in the specific activity over the crude preparation.
Adenosine, linked via its adenine-C
to agarose
(ag-adenosine, Pharmacia), has been successfully used to purify other
plant methyltransferases
(24, 25) . Our
adenosine-agarose matrix was highly stable and was repeatedly used
without any loss of its binding or eluting efficiencies. Other affinity
matrices prepared with either AdoMet or AdoHcy linked via their free
amino or carboxyl groups to Affi-Prep 10 (Bio-Rad) or EAH-Sepharose
(Pharmacia) failed to bind the enzyme.
Figure 4:
Competition between the alternative
substrates of the halide/bisulfide methyltransferase. A,
inhibition of iodide methylation by different fixed concentrations of
bisulfide. B, inhibition of bisulfide methylation by different
fixed concentrations of iodide. The concentration of AdoMet was kept
constant at 0.5 m
M. nkat,
nanokatals.
High performance liquid
chromatography on Protein Pak Q anion exchange column resolved multiple
peaks of halide/bisulfide methyltransferase activity (Fig. 3). An
enzyme preparation processed through a procedure in which salt
precipitation was replaced with polyethylene glycol precipitation also
gave multiple peaks in this anion exchange step (not shown). These
observations suggest that the enzyme may exist in multiple charge
isoforms, although this possibility remains to be confirmed through
further experiments. Interestingly, rat liver thiol methyltransferase,
with which the halide/bisulfide methyltransferase shares many
properties, was reported in five different cellular compartments
(26) . The harsh freeze-thaw treatment needed to maximize the
extraction of halide/bisulfide methyltransferase was also similar to
the procedure used to extract the compartmented rat liver enzyme
(18) .
Figure 3:
Elution of halide/bisulfide
methyltransferase from a Protein Pak Q column upon anion exchange high
performance liquid chromatography. Bound proteins were eluted in a
0-200 m
M and 200-500 m
M two-step linear
gradient of NaCl in buffer C. Halide- () and
bisulfide-methylating (
) activities were assayed in each
fraction. Proteins were monitored by absorbance at 280 nm ( solid line). nkat,
nanokatals.
The protein from a fraction containing peak
halide/bisulfide methyltransferase activity from the Superdex 75 step
migrated as a single band of 28,000 Da on SDS-PAGE (Fig. 1).
Moreover, when consecutive fractions across the first peak of activity
after anion exchange HPLC were applied to SDS-PAGE, the Coomassie Blue
staining intensity of the 28,000-Da protein band peaked in the fraction
containing peak enzyme activity (not shown). These observations
confirmed that the molecular mass of the denatured halide/bisulfide
methyltransferase was 28,000 Da. Since the molecular weight of the
enzyme, calculated from gel filtration chromatography, was 29,500, the
native protein probably functions as a monomer. The molecular weight of
this enzyme was similar to the algal methyl chloride transferase
(15) and mammalian thiol methyltransferase
(18, 26) .
ion concentration. Since H
S has a
p K
1 of 7.02
(27) ,
[HS
] would be expected to fall sharply as
the pH drops below 7. The halide- and bisulfide-methylating activities
had the same pI value at pH 4.8, which was consistent with the
observation that both activities existed on a single protein.
Figure 5:
Ef