Under these condi-tions, epithionitrile formation from allylglucosinolate was completely abolished, with allyl isothiocyanate being the only hydrolysis product formed data not shown.. If
Trang 1nitrile-forming proteins from plants and insects that alter myrosinase-catalysed hydrolysis of glucosinolates
Meike Burow, Jana Markert, Jonathan Gershenzon and Ute Wittstock
Max Planck Institute for Chemical Ecology, Department of Biochemistry, Jena, Germany
Plants defend themselves against herbivore and
patho-gen attack using a diverse array of repellent or toxic
secondary metabolites [1,2] Among these chemical
defences, the glucosinolates found in plants of the
order Capparales have been studied intensively as they
have significant effects on the taste, flavour, nutritional
value and pest resistance of crops belonging to the
Brassicaceae family, such as oilseed rape, cabbage and
broccoli [3,4] Glucosinolates are amino acid-derived
thioglycosides with aliphatic, aromatic or indole side chains (Fig 1) The biological activities of glucosino-late-containing plants are usually attributed to the hydrolysis products formed from glucosinolates upon tissue disruption by endogenous thioglucosidases (known as myrosinases EC 3.2.3.1., Fig 2) rather than
to the parent glucosinolates, which are spatially separ-ated from myrosinases in the intact plant [5,6] The most common type of glucosinolate hydrolysis type of
Keywords
epithionitrile; epithiospecifier protein;
glucosinolate; nitrile; nitrile-specifier protein
Correspondence
U Wittstock, Institut fu¨r Pharmazeutische
Biologie, Technische Universita¨t
Braunschweig, Mendelssohnstr 1,
D-38106 Baunschweig, Germany
Fax: +49 531 391 8104
Tel: +49 531 391 5681
E-mail: u.wittstock@tu-bs.de
(Received 1 March 2006, accepted
30 March 2006)
doi:10.1111/j.1742-4658.2006.05252.x
The defensive function of the glucosinolate–myrosinase system in plants of the order Capparales results from the formation of isothiocyanates when glucosinolates are hydrolysed by myrosinases upon tissue damage In some glucosinolate-containing plant species, as well as in the insect herbivore Pieris rapae, protein factors alter the outcome of myrosinase-catalysed glu-cosinolate hydrolysis, leading to the formation of products other than isothiocyanates To date, two such proteins have been identified at the molecular level, the epithiospecifier protein (ESP) from Arabidopsis thaliana and the nitrile-specifier protein (NSP) from P rapae These proteins share
no sequence similarity although they both promote the formation of nit-riles To understand the biochemical bases of nitrile formation, we com-pared some of the properties of these proteins using purified preparations
We show that both proteins appear to be true enzymes rather than
alloster-ic cofactors of myrosinases, based on their substrate and product specif-icities and the fact that the proportion of glucosinolates hydrolysed to nitriles does not remain constant when myrosinase activity varies No sta-ble association between ESP and myrosinase could be demonstrated during affinity chromatography, nevertheless some proximity of ESP to myrosin-ase is required for epithionitrile formation to occur, as evidenced by the lack of ESP activity when it was spatially separated from myrosinase in a dialysis chamber The significant difference in substrate- and product spe-cificities between A thaliana ESP and P rapae NSP is consonant with their different ecological functions Furthermore, ESP and NSP differ remark-ably in their requirements for metal ion cofactors We found no indications
of the involvement of a free radical mechanism in epithionitrile formation
by ESP as suggested in earlier reports
Abbreviations
BPDS, bathophenanthroline disulfonic acid; ESP, epithiospecifier protein; FID, flame ionization detection; NSP, nitrile-specifier protein.
Trang 2products, the isothiocyanates, has been shown to
pos-sess antimicrobial and insecticidal activities [7], and
have stimulated much interest as cancer-preventing
agents [8,9] In addition to isothiocyanates, other
hydrolysis products such as epithionitriles and
thiocya-nates are formed in other species of the Brassicaceae
under the influence of certain protein factors [10–12]
For example, epithiospecifier proteins (ESPs) have
been identified in several species of Brassicaceae that
alter the outcome of glucosinolate hydrolysis without
having hydrolytic activity on glucosinolates themselves
[13–15]
Since the first description of ESP activity in plants
in 1973 [13], only a few studies have investigated its
biochemical properties, probably because of the
diffi-culty in isolating the active protein from plant
mater-ial ESPs were originally described as 35–40 kDa proteins that promote the formation of epithionitriles, rather than isothiocyanates, from alkenylglucosinolates upon myrosinase-catalysed glucosinolate hydrolysis [12–14] During nitrile formation from alkenylglucosin-olates (Fig 1), the sulfur released from the thioglycosi-dic bond is captured by the terminal double bond in the glucosinolate side chain to form a thiirane (episul-fide) ring and an epithionitrile is formed [16] (Fig 2) However, the mechanism by which ESPs catalyse this intramolecular sulfur transfer is not known It has been suggested that the mechanism is analogous to the formation of epoxides catalysed by cytochrome P450-dependent monooxygenases [17] The first ESP gene was isolated several years ago from the model plant Arabidopsis thaliana ecotype Landsberg erecta (Ler) (Brassicaceae) [15] It encodes a 37-kDa protein (341 amino acids) with 45–55% amino acid sequence iden-tity to several A thaliana myrosinase-binding proteins
In the presence of myrosinase, crude extracts of Escherichia coli expressing recombinant A thaliana ESP catalysed the conversion of alkenylglucosinolates
to epithionitriles, as well as the conversion of nonalke-nylglucosinolates to simple nitriles lacking the thiirane ring [15] (Fig 2)
More recently, we identified a functionally related protein factor, designated a nitrile-specifier protein (NSP), in the midgut of larvae of the cabbage white butterfly, Pieris rapae [18] P rapae NSP cDNA encodes a polypeptide of 632 amino acids with a molecular mass of 73 kDa Like plant ESPs, P rapae NSP does not directly hydrolyse glucosinolates, but promotes the formation of nitriles rather than toxic
1
n C N S
S C
R
3 -O S O N
S R
aglycone
2
3 -SO O
c l G
N S
3
Fig 2 Glucosinolate hydrolysis Upon activation of the glucosinolate–myrosinase system, glucosinolates are hydrolysed by myrosinases yielding unstable aglycones These aglycones can then spontaneously undergo a Lossen rearrangement to form the corresponding isothio-cyanates (1) In several species of the Brassicaceae, ESPs promote the formation of epithionitriles (2) from aliphatic glucosinolates with a terminal double bond such as allylglucosinolate ESP from A thaliana, however, is capable of producing epithionitriles from alkenylglucosino-lates as well as simple nitriles (3) from nonalkenyl substrates Simple nitrile formation is also promoted by P rapae NSP or the presence of
Fe2+ Depending on the nature of the glucosinolate side chain, other hydrolysis products such as thiocyanates and oxazolidine-2-thiones can also be formed R indicates the variable side chain (aliphatic, aromatic or indole) of the parent glucosinolate.
S
N Glc
O SO 3
-S S
N
Glc
O SO 3
-S
N
Glc
O SO 3
-S
N Glc
O SO 3
-S O
2 1
4 3
Fig 1 Chemical structures of glucosinolates used in this study.
1, Allylglucosinolate; 2, 4-methylthiobutylglucosinolate; 3,
benzylglu-cosinolate; 4, 4-methylsulfinylbutylglucosinolate).
Trang 3isothiocyanates upon glucosinolate hydrolysis catalysed
by the myrosinases ingested with the plant tissue The
nitriles are excreted with the faeces [18] Although
nit-rile formation in both plants and their insect
herbiv-ores is accomplished through the action of protein
factors, the plant ESP and insect NSP do not show
any significant amino acid sequence similarity [18]
Thus, it is an open question if ESP and NSP share any
biochemical properties and whether nitrile formation
mediated by these two proteins occurs via the same
mechanism
Because ESP and NSP activities have been detected
only in association with myrosinase, it is not yet
known if these proteins act as cofactors of
myrosin-ase or possess catalytic activity of their own In the
case of ESP, it has been suggested that this protein
interacts with myrosinases in an allosteric manner
leading to conformational changes in the myrosinase
active site that modify the proportions of hydrolysis
products formed [14] Alternatively, both ESP and
NSP may possess catalytic activity and control the
outcome of glucosinolate hydrolysis by converting the
unstable aglycone intermediate released by
myrosin-ase, which typically rearranges to an isothiocyanate,
to a nitrile product instead In either case, it can be
assumed that ESP and NSP have to be closely
associ-ated with myrosinases, either to bind them as
cofac-tors or to bind the unstable aglycone before it
rearranges
The isolation of two different types of
nitrile-form-ing proteins, ESP from A thaliana and NSP from
P rapae, presented an opportunity to learn more
about the biochemical requirements for nitrile
forma-tion Here, we compare some characteristics of the
purified proteins to investigate their role in nitrile
for-mation We found striking differences between ESP
and NSP which are in agreement with their proposed
biological functions, but both appear to act as enzymes
rather than myrosinase cofactors Our data do not
support the mechanism for epithionitrile formation
suggested in an earlier report
Results
Development of an enzyme assay to measure
ESP-dependent nitrile formation
ESPs have been shown to redirect the hydrolysis of
glucosinolates catalysed by myrosinases from
isothio-cyanate formation towards formation of the
corres-ponding nitriles [10,13,15,17,19] The ability of ESPs
to promote nitrile formation in the presence of
myros-inase has been referred to as ESP activity even though
it is not yet certain if ESPs are cofactors rather than true enzymes Their activity has been detected only in conjunction with myrosinase If ESPs are catalysts, they can be assumed to convert the aglycones pro-duced by myrosinase to nitrile products The instability
of these aglycones precludes rigorous kinetic studies, and no other natural or synthetic compound has been reported to serve as a substrate for ESP
With these limitations in mind, optimal assay con-ditions for the biochemical characterization of the purified, recombinant A thaliana ESP were sought A purified preparation from Sinapis alba was used as the myrosinase source, and a number of buffers and
pH conditions were surveyed Surprisingly, ESP was completely inactive in citrate and phosphate buffers, but promoted nitrile formation in many biological buffers as measured by the production of epithiopro-pyl cyanide [2-(thiirane-2-yl)acetonitrile] from allyl glucosinolate (Figs 1,2) The highest activity was found in Mes buffer (50 mm) at pH 6.0 Fe2+ has been previously reported to be an essential cofactor for ESP from Brassica napus [17] and Crambe abyssi-nica [13] This was also true for our recombinant
A thaliana protein, with the optimal concentration found to be 0.5 mm The temperature for standard assays was chosen to be 20C Higher temperatures accelerated the hydrolysis of allyl glucosinolate, but the proportion of epithiopropyl cyanide declined rel-ative to the amounts of other hydrolysis products (Table 1)
Irrespective of whether ESP is a protein cofactor or
a true enzyme, the molar ratio of ESP to myrosinase can be assumed to be crucial for the proportion of nit-riles produced upon glucosinolate hydrolysis Under the standard conditions used for ESP assays, a 260-fold molar excess of ESP was employed relative to the quantity of myrosinase protein, which is a dimer Lower molar ratios of ESP:myrosinase resulted in decreased absolute and relative nitrile formation, as measured by the production of epithionitrile from allylglucosinolate When higher molar ratios of ESP to myrosinase were used, myrosinase activity was found
to be strongly reduced which reduced net nitrile forma-tion With a 260-fold molar excess of ESP:myrosinase, nitrile products and isothiocyanates each accounted for
50% of the hydrolysis products formed The produc-tion of considerable amounts of isothiocyanate indi-cates that ESP (assuming it acts as an enzyme) is saturated with its substrate The concentration of glucosinolates used in the assays (1–3 mm) was satur-ating with respect to myrosinase activity The same considerations were used in developing an assay for NSP activity
Trang 4Physical proximity of myrosinase and ESP is
essential for epithionitrile formation, but a stable
interaction could not be detected
Examination of the predicted structure of the A
thali-anaESP showed that most of the protein was made up
of a series of b sheets known as Kelch motifs [20]
(Fig 9), as predicted by the program interproscan
(http://www.ebi.ac.uk/InterProScan/ European
Bioin-formatics Institute, Hinxton, Cambridge, UK) These
features are known to mediate protein–protein
interac-tions providing some support for the hypothesis that
ESP functions as an allosteric cofactor of myrosinase
In this case, a very close physical interaction between
the two proteins would be expected To assess if an
association of ESP and myrosinase is essential for
epithionitrile formation, we spatially separated the two
proteins by placing ESP into a dialysis cassette with
myrosinase in the external buffer Under these
condi-tions, epithionitrile formation from allylglucosinolate
was completely abolished, with allyl isothiocyanate
being the only hydrolysis product formed (data not
shown) These results suggested that ESP and
myrosin-ase must have some proximity for nitrile formation to
occur To test for a stable association of ESP and my-rosinase, crude extracts of A thaliana (Col-0) leaves were added to an amino-link agarose resin coupled with an antibody to ESP that had been presaturated with ESP However, myrosinase activity was found only in the wash fractions containing unbound compo-nents of the plant extract When ESP was eluted from the resin, no myrosinase activity was detectable in the eluates (Fig 10) Furthermore, the recombinant ESP did not comigrate with the S alba myrosinase in native PAGE gels (data not shown)
ESP and NSP: enzymes or myrosinase cofactors? Another approach employed to investigate the role of ESP and NSP in nitrile formation was to manipulate the activity of myrosinase and observe its effect on nit-rile formation If ESP interacts with myrosinase as an allosteric cofactor, manipulation of myrosinase in the presence of ESP should only affect the total amount of hydrolysis products formed without altering the ratio between nitrile and isothiocyanate We stimulated myrosinase by addition of low concentrations of
l-ascorbate [21–23] l-Ascorbate facilitates release of the glucose moiety from the active site of myrosinases, the rate-limiting step in glucosinolate hydrolysis [21] The addition of 0.05–2 mm l-ascorbate to ESP assays increased the formation of total hydrolysis prod-ucts, as described previously, but decreased the amount
of epithionitrile formed from allylglucosinolate relative
to the amount of isothiocyanate (Fig 3A) A parallel series of assays carried out with the P rapae NSP gave similar results The increase in total hydrolysis products formed from benzylglucosinolate with l-ascorbate addi-tion was accompanied by a substantial decrease in the ratio of nitrile to isothiocyanate (Fig 3B)
ESP and NSP have different substrate and product specificities
To compare the properties of A thaliana ESP, purified after heterologous expression in E coli, and P rapae NSP, purified from larval midguts, we examined their effects on the myrosinase-catalysed hydrolysis of differ-ent aliphatic glucosinolates and the aromatic benzyl-glucosinolate (Fig 4, Table 2) When allylbenzyl-glucosinolate was incubated with myrosinase only, allyl isothiocya-nate was the major hydrolysis product ESP redirected the hydrolysis of allylglucosinolate towards the forma-tion of the corresponding epithionitrile, epithiopropyl cyanide [2-(thiirane-2-yl)acetonitrile] with minor amounts of the simple nitrile, allyl cyanide (but-3-ene nitrile) Both diastereomers of epithiopropyl cyanide
Table 1 Biochemical characteristics of ESP The effects of variable
temperature, phosphate ions, radical scavengers, and reducing
agents were tested on ESP-catalysed formation of the epithionitrile
from allylglucosinolate, epithiopropyl cyanide, in ESP assays
per-formed under standard conditions as described in Experimental
pro-cedures.
Test parameter Effect on ESP
Temperature Higher absolute and relative nitrile formation
at low temperatures (0–20 C) despite reduced myrosinase activity
Phosphate Complete inhibition with 5 m M phosphate
(Ki¼ 1 m M ); activity was restored by ferrous ions
Sulfate Acts as myrosinase inhibitor; addition of
sulfate (0–100 m M concentration tested) resulted in reduced absolute but constant relative activity
Sorbitol No effect (0–100 m M tested)
L -Cysteine No effect, but 5 m M L -cysteine led to a
decrease in myrosinase activity Superoxide
dismutase
No effect (5 lgÆassay)1)
Argon Flushing of assay mixtures with argon
had no effect Dithiothreitol Addition 0.1 m M dithiothreitol resulted in
a threefold increase in activity (only 0.5-fold increase in presence of 0.5 m M ferrous ions)
b-Mercaptoethanol 7 m M b-mercaptoethanol resulted in
a 20% increase in activity
Trang 5were detected by GC-MS using a chiral column for
separation (data not shown) The proportion of these
diastereomers was 1:1, suggesting that sulfur capture
by the terminal double bond is equally probable from
above or below When NSP was added to the assay,
hydrolysis was redirected only to the simple nitrile,
and no epithionitrile was formed (Fig 4)
The percentage of nitriles (relative to the total
amount of hydrolysis products) formed from different
glucosinolates in the presence of ESP varied depending
on the glucosinolate side chain ESP catalysed the formation of substantial amounts of nitriles from allyl-glucosinolate, 4-methylthiobutylglucosinolate and,
to a lesser extent, 4-methylsulfinylbutylglucosinolate However, almost no nitrile was produced from benzyl-glucosinolate under the same conditions The epithio-nitrile was the predominant epithio-nitrile formed from allylglucosinolate with small amounts of the simple nit-rile detected Only simple nitnit-riles were formed from the remaining glucosinolates In contrast, NSP promoted the formation of simple nitriles from allyl-, 4-methylthiobutyl-, 4-methylsulfinylbutyl- and benzyl-glucosinolate with comparable efficiencies No epithio-nitrile was seen to be formed from allylglucosinolate
In the absence of ESP or NSP, all four glucosinolates were hydrolysed almost exclusively to the correspond-ing isothiocyanates (Table 2, third row)
Fe2+promotes nitrile formation from different glucosinolates in the presence of ESP and NSP
Fe2+ has previously been reported to be essential for the catalytic activity of ESP from Brassica napus [17] and Crambe abyssinica [13] Thus the effects of Fe2+
on A thaliana ESP and P rapae NSP were investigated using different glucosinolates as substrates (Table 2) The addition of Fe2+to ESP assays increased the pro-portion of epithionitrile formed from allylglucosinolate from 27.0% (without addition of Fe2+) to 87.2% (0.01 mm Fe2+) and 93.9% (0.5 mm Fe2+) of the total amount of hydrolysis products No epithionitrile was formed by myrosinase in the presence of Fe2+without addition of ESP, indicating that ferrous ions and myrosinase by themselves are not sufficient for the for-mation of the thiirane ring In contrast, Fe2+promoted the formation of simple nitriles from the tested
gluco-Fig 3 Predicted protein structure of
A thaliana Ler ESP Amino acid sequence
of A thaliana ESP (GenBank accession num-ber AAL14623) and Kelch motif repeats as predicted by INTERPROSCAN Amino acids of the Kelch repeats are printed in bold, and the repeats are indicated by arrows Each Kelch repeat forms a b sheet (b1)5).
A
kDa L1 W1 L2 W2 E1 E2 E3
52
35
0 20 40 60 80 100
B
W2
Fig 4 Test for physical association between ESP and myrosinase.
A chromatography resin coupled with an anti-ESP serum was
satur-ated with ESP before it was loaded with a crude protein extract
from rosette leaves of A thaliana Col-0 containing myrosinase.
After a washing step, ESP and bound proteins were eluted
Load-ing, washes and elution were monitored by western blot analysis
with an anti-ESP serum (A) and by myrosinase assays (B) (L1,
flow-through after binding of ESP; W1, wash step after binding of
ESP; L2, flow-through after loading of plant extract containing
myrosinase; W2, wash step after loading of plant extract; E1–E3
eluted fractions of ESP and bound proteins)
Trang 6sinolates in the presence of myrosinase even without
ESP The addition of ESP further increased the
propor-tion of simple nitriles formed from 4-methylthiobutyl-,
4-methylsulfinylbutyl- and benzylglucosinolate In the
presence of 0.01 mm Fe2+, ESP caused an increase
in the formation of simple nitriles from
4-methyl-sulfinylbutyl- and to a lesser extent from
benzylglucosi-nolate compared with assays without ESP In the
presence of 0.5 mm Fe2+, ESP addition led to nearly
100% simple nitrile formation for all tested
nonalke-nylglucosinolates For NSP, addition of 0.01 mm Fe2+
resulted in an increase in simple nitrile formation from
allyl- and benzylglucosinolate compared with assays
without added Fe2+, but did not result in the
forma-tion of the epithionitrile from allylglucosinolate
Addition of Fe3+increases ESP activity but not
NSP activity
The activity of ESP measured in crude seed extracts of
Lepidium sativum has been shown to increase in the
presence of both Fe2+ and Fe3+ [24], whereas no
effect of Fe2+on the activity of ESP in Crambe
abyssi-nica seeds was found [25] To investigate whether or
not the impact of iron on the activity of the A
thali-ana ESP is restricted to Fe2+, assays were carried out
with both Fe2+ or Fe3+ (0.5 mm) Allylglucosinolate
was chosen as a substrate, because formation of the
corresponding epithionitrile is strictly dependent on
ESP and is therefore a better measure of ESP activity
than simple nitrile formation Saturation of ESP with
its substrate under standard assay conditions was
insured by reducing the amounts of ESP (0.3 units), such that the isothiocyanate was formed in consider-able amounts even under conditions of elevated ESP activity In this series of assays, the formation of epith-ionitrile accounted for a maximum of 85% of the amount of total hydrolysis products The results showed that addition of Fe2+in various forms resulted
in a 16-fold increase in ESP activity, while a sevenfold increase was observed for addition of Fe3+(Fig 5A)
To study the effects of Fe2+and Fe3+on nitrile for-mation by NSP, assays were carried out using benzyl-glucosinolate as substrate and adding iron salts to a final concentration of only 0.01 mm to avoid a high background of NSP-independent nitrile formation As described previously, the conversion of benzylglucosi-nolate to its corresponding simple nitrile by NSP was slightly increased by 0.01 mm Fe2+compared with no iron salt addition, but no changes in NSP activity were measured in the presence of Fe3+ (Fig 5B) Without the addition of ESP or NSP, the enhanced formation
of nitriles from myrosinase-catalysed hydrolysis of both allyl- and benzylglucosinolate was observed only upon addition of Fe2+, whereas Fe3+ did not change the proportion of nitriles produced by this reaction The anions of the iron salts used did not have any effects on ESP, NSP or myrosinase
ESP and NSP differ in their requirements for iron species
To study the role of iron in the catalytic mechanisms
of ESP and NSP in more detail, different chelators
Table 2 Nitrile and epithionitrile formation from different glucosinolates in the presence of myrosinase, ESP and NSP in vitro Assays were performed in 50 m M Mes, pH 6.0, containing 1 m M glucosinolate as substrate, with one exception: 3 m M 4-methylsulfinylbutylglucosinolate was used in ESP assays in order to compensate for the low activity of the Sinapis alba myrosinase with this substrate Ferrous ions were added as (NH4)2[Fe(SO4)2] to minimize oxidation Dichloromethane extracts of the assays were analysed by GC-MS and GC-FID The amounts of products are expressed as a percentage of the total amount of hydrolysis products measured in nmol In each case, the remain-der of the hydrolysis products were isothiocyanates Data are the mean ± SD of results obtained in at least three independent experiments 4-mtb-, 4-methylthiobutyl-; 4-msob-, 4-methylsulfinylbutyl-; CN, cyanide; myr, myrosinase; nd, not determined.
Glucosinolate Hydrolysis product allyl- epithio-propyl-CN allyl-CN 4-mtb- 4-mtb-CN 4-msob- 4-msob-CN benzyl- benzyl-CN
No Fe 2+ added
0.01 m M Fe2+
0.5 m M Fe 2+
Trang 7were added to the enzyme assays ESP activity was
strongly reduced in the presence of 5 mm
bathophen-anthroline disulfonic acid (BPDS), a chelator of
fer-rous ions, and by 5 mm deferoxamine, a chelator of
ferric ions (Fig 6A) The addition of 2 mm EDTA,
known to chelate Fe2+and Fe3+, as well as numerous
other cations, was sufficient to completely inhibit
ESP-dependent epithionitrile formation in favour of the
generation of isothiocyanate (Fig 7) In assays
per-formed using 5 mm EDTA, ESP activity was restored
by addition of 0.5 mm Fe2+ or 0.5 mm Fe3+
(Fig 8A), whereas Ca2+, Mg2+and K+had no effect
However, when Ca2+ or Mg2+ was added to a final
concentration of 5 mm, the epithionitrile of
allylglucos-inolate was formed (data not shown)
The activity of NSP, measured by nitrile formation
from benzylglucosinolate, was changed only by
chela-tors that complex divalent cations Addition of EDTA and BPDS reduced the proportion of nitrile formed from 36.3 to 16.9 and 15.0%, respectively (Fig 6B)
In contrast, deferoxamine did not affect NSP activity,
A
0
20
40
60
80
100
120
140
160
L-ascorbate [m M ]
B
0
20
40
60
80
100
120
140
160
L-ascorbate [m M ]
Fig 5 Effects of L -ascorbate on ESP and NSP (A) ESP assays
were carried out in 50 m M Mes, pH 6.0, containing 1 m M
allylglu-cosinolate, 0.3 units ESP and 2 units myrosinase (grey,
epithiopro-pyl cyanide; white, allyl isothiocyanate) (B) NSP assays were
performed under the same conditions using benzylglucosinolate,
1 unit NSP and 1 unit myrosinase (grey, benzyl cyanide; white,
ben-zyl isothiocyanate) Data are the mean ± SD of three independent
experiments.
A
S
NCS CN
3
0 200
400
IS
2 1
B
IS
2 1
400
200
0
12
6
C
IS
2
400
200
0
12
D
retention time [min]
Fig 6 The effects of ESP and NSP on myrosinase-catalysed hydro-lysis of allylglucosinolate (A) Chemical structures of the hydrohydro-lysis products of allylglucosinolate: 1, allyl cyanide; 2, allyl isothiocyanate;
3, epithiopropyl cyanide (two diastereomers) (B–D) Allylglucosino-late was incubated with ESP and myrosinase (B), NSP and myrosin-ase (C), or only myrosinmyrosin-ase (D) Assays were performed in 50 m M Mes, pH 6.0, containing 1 m M allylglucosinolate Shown are GC-FID chromatograms of dichloromethane extracts (IS, internal standard) Compound 3 was present as a 1:1 mixture of two diastereomers
as shown, which were separated only by gas chromatography using a chiral column (data not shown).
Trang 8indicating that Fe3+ does not play a major role in
NSP-catalysed nitrile formation The reduction in NSP
activity observed in the presence of 5 mm EDTA
was compensated for by addition of 0.5 mm Fe2+
(Fig 8B) However, a slight increase in nitrile
forma-tion by NSP was also seen upon the addiforma-tion of Fe3+
(0.5 mm) in the presence of EDTA (5 mm) This result
can be explained by displacement of small amounts of
ferrous ions from EDTA-binding sites due to the
smal-ler equilibrium dissociation constant of the Fe3 ±
EDTA complex
Further biochemical characterization of ESP
provides no support for any established
reaction mechanism
It has been suggested that ESP-catalysed epithionitrile
formation is similar to Fe-dependent epoxidations
mediated by cytochrome P450-dependent
monooxygen-ases [17], which are oxygen-requiring catalysts that
employ a free-radical mechanism To test this
proposi-tion, reactions were carried out in the absence of
oxy-gen or with radical scavenging aoxy-gents However, no
changes in ESP activity were measured in assays
per-formed with oxygen excluded using an argon purge
(Table 1) Addition of sorbitol, l-cysteine or
superox-ide dismutase as radical scavengers also did not affect
the absolute or relative amounts of epithionitrile formed by ESP
To investigate whether sulfhydryl groups play a role
in the mechanism of epithionitrile formation, ESP assays were carried out in the presence of dithiothreitol and b-mercaptoethanol Addition of dithiothreitol to a final concentration of 0.1 mm resulted in a threefold increase in the formation of epithionitrile from allyl-glucosinolate However, in the presence of 7 mm b-mercaptoethanol, only a 20% increase in ESP activ-ity was detected
Discussion
The outcome of myrosinase-catalysed glucosinolate hydrolysis can be altered by plant proteins that have
no hydrolytic activity on glucosinolates themselves Several species of the Brassicaceae including A thali-ana have been shown to produce epithionitriles and simple nitriles upon tissue damage instead of isothiocyanates due to the presence of an ESP [12,13,15,16] In this study, we characterized the ESP from A thaliana, ecotype Ler, in order to learn more about the mechanism by which this protein controls the outcome of glucosinolate hydrolysis We compared the biochemical properties of the A thaliana ESP with those of the functionally related NSP from the midgut
A
no Fe added 0
20 40 60 80 100 120 140
40 35 30 25 20 15 10
0 5
FeCl3
Fe2(SO4)3 FeCl2
(NH4)2[Fe(SO4)2] FeSO4
FeCl3
Fe2(SO4)3 FeCl2
(NH4)2[Fe(SO4)2] FeSO4
no Fe added
B
Fig 7 Effects of iron salts on ESP and
NSP (A) ESP activity was measured in
50 m M Mes, pH 6.0, containing 0.3 units
ESP, 1 m M allylglucosinolate and 2 units
myrosinase Salts were added to a final
con-centration of 0.5 m M For each salt, the left
bar represents the hydrolysis products
formed in the presence of ESP (dark green:
epithiopropyl cyanide; brown: allyl cyanide;
light green: allyl isothiocyanate), while the
right bar represents the products formed by
myrosinase in the absence of ESP (dark
grey, allyl cyanide; light grey, allyl
isothiocya-nate) (B) NSP assays were carried out
under the same conditions using 1 unit
NSP, 1 unit myrosinase and 1 m M
benzylglu-cosinolate as substrate Salts were added to
a final concentration of 0.01 m M For each
salt, the left bar represents the hydrolysis
products formed in the presence of NSP
(brown: benzyl cyanide; light green: benzyl
isothiocyanate), while the right bar
repre-sents the products formed by myrosinase in
the absence of NSP (dark grey, benzyl
cyan-ide; light grey, benzyl isothiocyanate) Data
are the mean ± SD of results obtained in
three independent experiments.
Trang 9of larvae of P rapae, a protein factor that redirects
the myrosinase-catalysed hydrolysis of glucosinolates
in ingested plant material towards the formation of
nitriles instead of toxic isothiocyanates [18] Studies
were carried out using purified recombinant A thaliana
ESP, P rapae NSP purified from larval midgut
extracts, and a pure preparation of myrosinase isolated
from seeds of S alba The use of purified enzymes
enabled us to perform all enzyme assays under strictly
defined conditions It has been suggested that plant
ESPs are not enzymes, but rather allosteric protein
co-factors that bind to myrosinases and change their
product specificities thereby promoting the formation
of epithionitriles and simple nitriles upon glucosinolate
hydrolysis [14] This proposal is supported by the lack
of stereospecificity in epithionitrile formation seen here
and in previous studies [26] In addition, the predicted
structure of the A thaliana ESP protein contains Kelch motifs (Fig 9) that are known to mediate protein– protein interactions However, our results did not con-firm a stable interaction between ESP and myrosinase during chromatographic separation (Fig 10) In addi-tion, the A thaliana ESP migrated separately from myrosinase on native PAGE gels (data not shown) These findings are in agreement with earlier reports on the separation of ESP and myrosinase from Crambe abyssinica and Brassica napus by gel filtration chroma-tography [13,19] Nevertheless, some proximity of ESP
to myrosinase seems to be required for epithionitrile formation because no ESP activity was detected when the two proteins were spatially separated by a dialysis membrane
To investigate further whether ESP acts as cofactor
of myrosinase or a separate enzyme, the ratio of epith-ionitrile to isothiocyanate formed from allylglucosino-late in the presence of the A thaliana ESP or the
P rapae NSP was monitored as myrosinase activity was increased by the addition of l-ascorbate [21–23] For both ESP and NSP, the ratios of hydrolysis prod-ucts changed markedly with an increase in myrosinase activity (Fig 3) These results do not support a role for NSP and ESP as myrosinase cofactors, but are more consistent with a catalytic role for these nitrile-forming proteins in which the unstable product of the myrosinase reaction serves as a direct substrate for nitrile formation In this interpretation, ESP and NSP become quickly saturated as the activity of myrosinase
is increased upon ascorbate addition, and the excess myrosinase product then rearranges to form additional isothiocyanate A catalytic function for ESP and NSP
A
Def.
BPDS EDTA
no addition
50
40
30
20
10
0
B
50
40
30
20
10
0
no addition
EDTA BPDS Def.
Fig 8 Effects of metal ion chelators on ESP and NSP (A) ESP
activity was assayed in 50 m M Mes, pH 6.0, containing 1 m M
allyl-glucosinolate, 3 units ESP and 4 units myrosinase (B) NSP assays
were performed under the same conditions using 1 unit NSP and
1 unit myrosinase Chelators were added to a final concentration of
5 m M Activities of ESP and NSP are expressed as the amount of
epithiopropyl cyanide (ESP) and benzyl cyanide (NSP) as a
percent-age of the total amount of hydrolysis products measured in nmol.
Data are the mean ± SD of results obtained in three independent
experiments BPDS, bathaphenanthroline disulfonic acid; Def.,
defe-roxamine; -CN, cyanide.
0 50 100 150 200 250
10
EDTA [mM]
Fig 9 ESP activity in the presence of EDTA Assays were per-formed under standard ESP assay conditions using three units ESP and allylglucosinolate as a substrate All products formed are given
in nmol (m, epithiopropyl cyanide; h, allyl isothiocyanate; s, allyl cyanide) All data are the mean ± SD from results obtained in three independent experiments.
Trang 10is also supported by the pronounced substrate
specifici-ty of ESP (Table 2) and the increased ESP activispecifici-ty
observed in conjunction with reduced myrosinase
activity at lower temperatures (Table 1)
Because the putative substrates of ESP and NSP
are highly unstable, there is currently no way of
assaying ESP and NSP activity other than in
com-bined assays in which the substrates for ESP and
NSP are delivered by myrosinase through hydrolysis
of the corresponding glucosinolates Although this
precludes rigorous determination of their kinetic
parameters, we compared a number of properties of
ESP and NSP and found some fundamental differ-ences between these two nitrile-forming proteins Under natural conditions, both ESP and NSP encounter a complex mixture of glucosinolates with variable side chains Therefore, we investigated the influence of ESP and NSP on the myrosinase-cata-lysed hydrolysis of different parent glucosinolates Glucosinolates without alkene function in their side chains were converted to simple nitriles by both pro-teins However, when allylglucosinolate was used as a substrate, ESP promoted the formation of the corres-ponding epithionitrile, but the simple nitrile, allyl cyanide (¼ but-3-ene nitrile) was not formed in signi-ficant amounts with ESP under any assay conditions tested In the presence of NSP, only the simple
nitri-le, and not the epithionitrinitri-le, was formed from al-lylglucosinolate (Fig 4) This major difference in product spectrum between ESP and NSP may reflect
a fundamental difference in their reaction mecha-nisms
Among different ecotypes of A thaliana, the pres-ence of a functional ESP was found to coincide with the accumulation of alkenylglucosinolates [15] ESPs might therefore have a special role in plants in the formation of epithionitriles, a class of glucosinolate hydrolysis products that could be an effective defence against insect herbivores due to the reactive thiirane ring When ESP assays were carried out using the nonalkenyl aliphatic glucosinolates, 4-methylthiobutyl-and 4-methylsulfinylbutylglucosinolate, the corres-ponding simple nitriles were formed However, the aromatic benzylglucosinolate was mainly hydrolysed
to benzyl isothiocyanate under the same conditions (Table 2) This distinct substrate specificity of
A thaliana ESP suggests that the function of this pro-tein lies in the formation of specific nitrile hydrolysis products rather than an overall decrease in isothio-cyanate formation In contrast, the function of NSP from P rapae may be to prevent the general forma-tion of isothiocyanates, which have been shown to reduce the survival and the growth of these insect herbivores [27] In vitro, P rapae NSP promotes
nitri-le formation from all glucosinolates tested with com-parable efficiencies (Table 2) indicating that the hydrolysis of any glucosinolate present in the larval host plants can probably be redirected towards nitrile formation
To compare the mechanism of epithionitrile and simple nitrile formation by the plant ESP with the mechanism of simple nitrile formation catalysed by the insect NSP, we studied some of the biochemical prop-erties of these two proteins Plant ESPs from differ-ent sources have previously been described as
A
B
45
40
35
30
25
20
15
10
5
0
no EDTA no
metal ion
Fe 2+ Fe 3+ Ca 2+ Mg 2+ K +
no EDTA no
metal ion
Fe 2+ Fe 3+ Ca 2+ Mg 2+ K +
45
40
35
30
25
20
15
10
5
0
Fig 10 Effects of metal ions on ESP and NSP in the presence of
EDTA (A) ESP assays were carried out in 50 m M Mes, pH 6.0,
containing 1 m M allylglucosinolate, 1 unit ESP and 4 units
myrosin-ase (B) The activity of 1 unit NSP was measured as nitrile
form-ation from benzylglucosinolate in the presence of 1 unit myrosinase.
EDTA was used at a concentration of 5 m M Salts of metal ions
[(NH4)2Fe(SO4)2, NH4Fe(SO4)2CaCl2, MgCl2, KCl] were added to a
final concentration of 0.5 m M Activities of ESP and NSP are
expressed as the amount of nitrile formed as a percentage of the
total amount of hydrolysis products measured in nmol Data are the
mean ± SD from three independent experiments.