Báo cáo khoa học: Brassinin oxidase, a fungal detoxifying enzyme to overcome a plant defense – purification, characterization and inhibition potx

Brassinin oxidase, a fungal detoxifying enzyme to
overcome a plant defense – purification, characterization
and inhibition
M. S. C. Pedras, Zoran Minic and Mukund Jha
Department of Chemistry, University of Saskatchewan, Canada
Microbial plant pathogens display a variety of succe-
ssful strategies to invade plant tissues and obtain the
necessary nutrients that allow growth and reproduc-
tion. Plants fight back with no smaller a variety of
weapons, including the synthesis of small to very large
molecules to inhibit specific metabolic processes in the
pathogen [1–3]. In general, plants under microbial
attack produce de novo a blend of antimicrobial
defenses known as phytoalexins, the specific compo-
nents of which appear to depend on the type of stress
[4,5]. Despite such an arsenal, fungal pathogens can
disarm the plant by counterattacking with enzymes
that detoxify promptly these phytoalexins [6–8]. The
outcome of this ‘arms race’ [3] frequently favors the
pathogen, causing great crop devastation and substan-
tial yield losses. Brassinin is a phytoalexin of great
importance to crucifer plants, due to its dual role both
as an antimicrobial defense and a biosynthetic precur-
sor of several other phytoalexins. The toxophore group
of brassinin is a dithiocarbamate, with an interesting
resemblance to the potent fungicides used in the 1960s
[9]. From a plant’s perspective, it is highly desirable to
prevent brassinin detoxification by any pathogen.
Crucifers include a wide variety of crops cultivated
across the world; for example, the oilseeds rapeseed
and canola (Brassica napus and Brassica rapa) and

vegetables such as cabbage (Brassica oleraceae var.
capitata), cauliflower (Brassica oleraceae var. botrytis)
or broccoli (Brassica oleraceae var. italica). In addi-
tion, both wild and cultivated crucifers are known to
Keywords
brassinin oxidase; camalexin; detoxifying
enzyme; Leptosphaeria maculans;
phytoalexin
Correspondence
M. S. C. Pedras, Department of Chemistry,
University of Saskatchewan, 110 Science
Place, Saskatoon, Saskatchewan S7N 5C9,
Canada
Fax: +1 306 966 4730
Tel: +1 306 966 4772
E-mail:
(Recived 24 April 2008, revised 17 May
2008, accepted 21 May 2008)
doi:10.1111/j.1742-4658.2008.06513.x
Blackleg fungi [Leptosphaeria maculans (asexual stage Phoma lingam) and
Leptosphaeria biglobosa] are devastating plant pathogens with well-estab-
lished stratagems to invade crucifers, including the production of enzymes
that detoxify plant defenses such as phytoalexins. The significant roles of
brassinin, both as a potent crucifer phytoalexin and a biosynthetic precur-
sor of several other plant defenses, make it critical to plant fitness. Brassi-
nin oxidase, a detoxifying enzyme produced by L. maculans both in vitro
and in planta, catalyzes the detoxification of brassinin by the unusual oxi-
dative transformation of a dithiocarbamate to an aldehyde. Purified brassi-
nin oxidase has an apparent molecular mass of 57 kDa, is approximately
20% glycosylated, and accepts a wide range of cofactors, including quinon-

es and flavins. Purified brassinin oxidase was used to screen a library of
brassinin analogues and crucifer phytoalexins for potential inhibitory activ-
ity. Unexpectedly, it was determined that the crucifer phytoalexins cama-
lexin and cyclobrassinin are competitive inhibitors of brassinin oxidase.
This discovery suggests that camalexin could protect crucifers from attacks
by L. maculans because camalexin is not metabolized by this pathogen and
is a strong mycelial growth inhibitor.
Abbreviations
BO, brassinin oxidase; CKX, cytokinin oxidase ⁄ dehydrogenase; DEA, diethanolamine; FCC, flash column chromatography; PMS, phenazine
methosulfate; PNGase, N-glycosidase; Q
0,
2,3-dimethoxy-5-methyl-1,4-benzoquinone; Q
10,
2,3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone.
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3691
have positive effects on human health (e.g. a high
intake of crucifers is associated with a reduced risk of
cancer) [10]. Economically significant diseases of cru-
cifer oilseeds and vegetables caused by fungi such as
the ‘blackleg’ fungi [Leptosphaeria maculans (asexual
stage Phoma lingam) and Leptosphaeria biglobosa] are
a global issue [11]. L. maculans is a pathogen with
well-established stratagems to invade crucifers, includ-
ing the production of enzymes that detoxify essential
phytoalexins [7]. For example, the phytoalexin brassi-
nin is detoxified via oxidation to indole-3-carboxalde-
hyde [7] or hydrolysis to indolyl-3-methanamine
(Fig. 1) [12].
Considering the apparent specificity of the enzyme
involved in the oxidative detoxification of brassinin,

brassinin oxidase (BO), we suggested that BO inhi-
bitors could prevent detoxification of brassinin by
L. maculans and thus avoid its depletion in infected
plants [13,14]. The concomitant accumulation of brass-
inin and related phytoalexins might prompt a recovery
in which the infected plant would be able to ward off
the sensitive pathogen(s). To better understand the role
of BO and test potential inhibitors, the enzyme was
purified, characterized and shown to be a novel
enzyme, consistent with the unusual transformation it
catalyzes (Fig. 1). Purified BO was used to screen a
library of 78 compounds containing crucifer phytoal-
exins and analogues for potential inhibitory activity.
Surprisingly, we determined that the crucifer phytoal-
exins camalexin and cyclobrassinin inhibited BO activ-
ity substantially but BO activity was not affected by
most of the synthetic compounds. This discovery
suggests that, if camalexin was co-produced with brass-
inin [5], it might protect Brassica sp. from attacks by
L. maculans because camalexin is not metabolized
by this pathogen and is a strong mycelial growth
inhibitor.
Results
Purification of BO activity
Fungal cultures initiated from spores were grown
under standard conditions and crude cell-free homo-
genates were prepared from mycelia, as reported in the
Experimental procedures. The enzyme was purified by
monitoring BO activity using brassinin as substrate.
Table 1 indicates the degree of purification and yield

obtained for each step. This purification protocol
involved four steps: first employing DEAE-Sephacel,
followed by chromatofocusing with PBE resin, then
Superdex 200 and, finally, Q-Sepharose chromato-
graphy. Fractions with BO activity obtained in the last
chromatography column were pooled, concentrated
and used for biochemical analysis. The purity of the
protein isolated after Q-Sepharose chromatography
was examined by SDS ⁄ PAGE, which, upon staining
with Coomassie brilliant blue R-250, revealed only one
band having the apparent molecular mass of 57 kDa
(Fig. 2). In addition, Superdex 200 chromatography of
the purified protein suggested that it was a native
monomer because it was eluted at a position corre-
sponding to a molecular mass similar to that deter-
mined by SDS ⁄ PAGE.
Fig. 1. Detoxification of the phytoalexin
brassinin by the ‘blackleg’ fungi L. maculans
(L. m.) and L. biglobosa (L. b.).
Table 1. Enzyme yields and purification factors for BO. Recoveries are expressed as a percentage of initial activity and purification factors
are calculated on the basis of specific activities (lmolÆmin
)1
= U).
Purification step
Yield
Specific
activity
(mUÆmg
)1
)

Recovery
(%)
Purification
factor (fold)
Protein (mg) Activity (mU)
Crude homogenate
a
120 187 1.6 100 1
DEAE-Sephacel 11 164 15 88 10
Chromatofocusing 0.59 82 139 44 89
Superdex 200 0.025 16 640 9 410
Q Sepharose 0.014 12 857 6 549
a
Mycelia from 1 L cultures yielded approximately 120 mg of protein.
Brassinin oxidase, a fungal detoxifying enzyme M. S. C. Pedras et al.
3692 FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS
Cellular localization of BO
The cellular localization of BO isolated from L. macu-
lans was established after fractionation of the crude
protein extract into soluble, membrane and cell wall
fractions, and these fractions were used for enzymatic
assays. As shown in Table 2, the BO specific activity
was found to be the highest in the cell wall fraction,
suggesting that BO was secreted (i.e. a cell wall pro-
tein). However, the total BO activity was found to be
the highest in the soluble fraction, which could imply
that this protein was present in the cytoplasm as well.
Because these inconclusive results were likely due to
contamination of the soluble protein fraction with cell
wall proteins, an additional fractionation was carried

out using concanavalin A chromatography [15,16], a
lectin used for purification of glycoproteins [17,18].
The majority of secreted proteins are glycosylated and
thus bind lectins specifically, namely those containing
mannose or glucose (e.g. concanavalin A) [19–21].
Hence, the protein extracts of both soluble and cell
wall fractions were subjected to concanavalin A Sepha-
rose chromatography. A single peak of activity was
obtained after eluting each column with methyl-a-d-
glucopyranoside (see supplementary Fig. S1). Similar
results were obtained using protein extracts from the
first purification step using DEAE-Sephacel chroma-
tography. The maximum enzyme recovery was
obtained using a relatively high concentration of
methyl-a-d-glucopyranoside (1.0 m). These results
suggest that BO is glycosylated and likely localized in
the cell wall.
Analysis of deglycosylated BO
The cellular localization assays and the ability of BO
to bind concanavalin A suggested that BO was an
N-glycosylated protein. To determine whether BO is
indeed a glycoprotein, purified BO was subjected to
treatment with N-glycosidase (PNGase) F, an enzyme
that cleaves N-linked oligosaccharides from proteins.
SDS ⁄ PAGE analysis showed a shift in the migration
of BO (46 kDa) in the sample treated with PNGase
versus the untreated sample (57 kDa) (Fig. 2A), dem-
onstrating that BO is an N-glycosylated protein
(approximately 20%). To further characterize the nat-
ure of the N-glycosylation of BO, samples of purified

BO were treated with endo-b-N-acetylglucosaminidase,
an enzyme that cleaves all high-mannose
oligosaccharides from proteins. Purified BO treated
with endo-b-N-acetylglucosaminidase (Fig. 2B) also
showed a shift in the migration of BO (47 kDa) com-
pared with the untreated sample (57 kDa) (Fig. 2B).
Identification of BO tryptic peptides by
LC-ESI-MS ⁄ MS
Glycoproteins can escape analysis at any level of a
peptide mass mapping procedure, in particular, during
tryptic digestion, due to potential steric disturbance
through interaction of the protein with proteolytic sites
of trypsin [22]. For this reason, to determine the pep-
tide sequence, analysis was performed with purified BO
after treatment with PNGase F. The deglycosylated
BO band in Fig. 3 was digested with trypsin and then
analyzed by LC-MS ⁄ MS using mascot algorithms. In
total, 20 peptides were deduced from the LC-MS ⁄ MS
spectral data (Table 3). The sequence homology of the
identified peptides was analyzed using the NCBI blast
algorithm. Peptides did not match significantly
with proteins available in the NCBI blast database.
Fig. 2. SDS ⁄ PAGE of protein fractions from purification of BO.
Lane M, marker proteins (molecular masses are indicated); lane 1,
crude homogenate (40 lg); lane 2, DEAE-Sephacel pooled fractions
(10 lg); lane 3, Polybuffer exchanger 94 chromatography (10 lg);
lane 4, Superdex 200-pooled fractions (1.5 lg); lane 5, purified BO
after Q-Sepharose chromatography (1 lg).
Table 2. Fractionation of proteins from L. maculans for cellular
localization of BO.

Protein
fraction
Volume
(mL)
Protein
(mg)
Specific
activity
(nmolÆmin
)1
Æmg
)1
)
Total
activity
(nmolÆmin
)1
)
Soluble 20 50 1.31 65
Cell wall 10 10 1.70 17
Membrane 6 59 0.21 12
M. S. C. Pedras et al. Brassinin oxidase, a fungal detoxifying enzyme
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3693
However, analysis of peptides using NCBI blast data-
base pertaining to fungi revealed that the majority of
peptides in Table 3 had some homology to a putative
short-chain dehydrogenase from Aspergillus terreus
NIH2624 (accession no. XP_001210968) and putative
NADP-dependent flavin oxidoreductase from Asper-
gillus nidulans FGSC A4 (accession no. XP_663310)

(results not shown).
Characterization of BO
BO required the presence of an electron acceptor for
activity. The purified enzyme was examined in the
presence of various electron acceptors at concentra-
tions of 0.10 and 0.50 mm. As shown in Table 4, BO
could accept a wide range of cofactors, including phen-
azine methosulfate (PMS), 1,4-benzoquinone, 1,2-
naphthoquinone, 2,6-dichloroindophenol;, coenzyme
2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q
0
) and
FMN. The highest BO activity was obtained with PMS.
The quinones 1,4-antraquinone and coenzyme 2,3-meth-
oxy-5-methyl-6-geranyl-1,4-benzoquinone (Q
10
) were
not accepted, whereas the flavin derivative FMN acted
as an electron acceptor. A number of other electron
acceptors, such as FAD, duraquinone, NADP, cyto-
chrome c and CuCl
2
, had low or no detectable effect
on BO activity. The absorbance spectrum of purified
BO (0.1 mgÆmL
)1
) revealed a peak at 280 nm, typical
of proteins containing aromatic amino acids, but no
chromophores characteristic of flavin or quinone
dependant oxidoreductases were detected (no absorp-

tion observed in the range 300–600 nm; results not
shown).
The kinetic parameters for BO activity were deter-
mined using brassinin as substrate in the presence of
PMS as an electron acceptor. Substrate saturation
curves were fitted to the Michaelis–Menten equation to
obtain the kinetic parameters. The apparent K
m
and
k
cat
were 0.15 mm and 0.95 s
)1
, respectively. The cata-
lytic efficiency (k
cat
⁄ K
m
) was determined to be of
6333 s
)1
Æm
)1
. The apparent K
m
for PMS was 0.30 lm.
The influence of pH on the activity of the BO was
investigated in the range pH 3–11. The pH optima
were determined to be in the range 8.0–10.0 (results
not shown). The temperature dependence of BO activ-

ity was tested in the range 8–75 °C, and the apparent
optimum temperature was 45 °C (results not shown).
Identification of inhibitors of BO
Several analogues of brassinin and phytoalexins (78
compounds; see supplementary Table S1) were synthe-
sized, purified and characterized spectroscopically, as
reported previously [13,14]. The activity of BO was
examined in the presence of these compounds at
0.10 mm (supplementary Table S1); the compounds
showing inhibition were also tested at 0.30 mm
(Table 5). Camalexin, cyclobrassinin, thiabendazole
and isobrassinin inhibited BO activity, whereas none
of the remaining compounds had an effect. Further-
more, none of the compounds shown in supplementary
Table S1 were substrates of BO. Considering the
A
B
Fig. 3. SDS ⁄ PAGE of deglycosylated BO. Purified BO was incu-
bated with and without (A) PNGase F and (B) endo-b-N-acetyl-
glucosaminidase as described in the Experimental procedures.
Deglycosylated samples were separated by SDS ⁄ PAGE and migra-
tion of deglycosylated BO was estimated by comparison with molec-
ular markers. (A) Overnight incubation of BO in nondenaturing
conditions with PNGase F results in a reduction of molecular mass
of BO (46 kDa) compared with nontreated BO (57 kDa). Treatment
of BO with PNGase F in denaturing conditions for 3 h also results in
a reduction of molecular mass of BO (46 kDa) compared with non-
treated BO (57 kDa). (B) Endo-b-N-acetylglucosaminidase treatment
of BO in denaturing conditions for 3 h results in a reduction of mole-
cular mass of BO (47 kDa) compared with nontreated BO (57 kDa).

Brassinin oxidase, a fungal detoxifying enzyme M. S. C. Pedras et al.
3694 FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS
substantially higher inhibitory effect of both camalexin
and cyclobrassinin, it was of great importance to deter-
mine the type of inhibition that each compound
displayed. The kinetics of inhibition of BO is shown in
the form of Lineweaver–Burk double reciprocal plots
(1 ⁄ S versus 1 ⁄ V) using 0.10 and 0.30 mm concentra-
tions of camalexin and cyclobrassinin (Fig. 4). The
results showed that the intersection points of all curves
were on the 1 ⁄ V axis (i.e. both camalexin and cyclo-
brassinin competitively inhibited BO activity).
Kinetic mechanism of BO
The bisubstrate reaction mechanism of BO involves
the oxidation of brassinin by an electron acceptor such
as PMS. Steady-state kinetic studies were performed to
Table 3. Masses and scores of tryptic peptides
a
obtained from deglycosylated BO after treatment by PNGase. Observed, mass ⁄ charge of
observed peptide; M
r
(expt), observed mass of peptide; M
r
(calc), calculated mass of matched peptide; Delta, difference (error) between the
experimental and calculated masses; Score, ions score. A score of 49 or greater indicates that the probability of an incorrect match is < 5%.
Observed M
r
(expt) M
r
(calc) Delta Score Peptide

442.2943 882.5741 882.3865 0.1876 33 QSSASTMR
453.2606 904.5066 904.4766 0.030 59 KALAAFAADRA
453.2606 904.5066 904.5130 –0.0064 67 RLAAAFAVSRM
473.2829 944.5512 944.5444 0.0068 34 RAVFPSIVGRS
521.2741 1040.5336 1040.5079 0.0257 33 AYPGYAPFR
566.7698 1131.5249 1131.5197 0.0053 36 RGYSFTTTAERE
585.3552 1168.6958 1168.6928 0.0030 57 RNTLLIAGLQARN
621.3502 1240.6858 1240.7074 –0.0216 26 MLLLSQPGRAR
656.7603 1311.5061 1311.5765 –0.0704 21 TLYGGMLDDDGR
708.8882 1415.7619 1415.7660 –0.0041 73 KDQLLLGPTYATPKV
710.3819 1418.7492 1418.7405 0.0087 90 RLEGLTDEINFLRQ
797.9465 1593.8784 1593.9315 –0.0531 15 LAAPVAVVTGASRGIGR
544.2526 1629.7358 1629.7132 0.0227 59 KHSGPNSADSANDGFVRL
585.9861 1754.9364 1754.9277 0.0087 46 RGMGGAFVLVLYDEIKKF
626.6251 1876.8536 1876.8520 0.0016 77 KNASCTLSSAVHSQCVTRL
635.9510 1904.8311 1904.9513 –0.1202 20 VVSESNQATNLLTAEMKA
1005.9999 2009.9852 2009.9807 0.0046 96 KVSGAAAQQAVSYPDNLTYRD
729.6232 2185.8478 2185.9626 –0.1148 20 GYYAMDYWGQGTSVTVSSAK
761.3307 2280.9703 2281.1087 –0.1384 137 RDAAVSPDLGAGGDAPAPAPAPAHTRD
872.7958 2615.3655 2615.3411 0.0244 89 DVLMTRTPLSLPVSLGDQASISCRS
Table 4. Effect of electron acceptors on BO activity. BO activities
measured under standard assay conditions described in the Experi-
mental procedures; results are expressed as the means ± SD of
three independent experiments; relative activity is expressed as
percentage of the reaction rate obtained with PMS. ND, not
detected.
Cofactor (electron acceptor)
Relative activity (%)
0.10 m
M 0.50 mM

PMS 94 ± 2 100
a
1,4-Benzoquinone 66 ± 4 77 ± 6
1,2-Naphthoquinone 57 ± 8 75 ± 19
2,6-Dichloroindophenol; 61 ± 9 62 ± 6
Coenzyme Q
0
47 ± 2 62 ± 3
FMN 36 ± 9 59 ± 12
K
3
[Fe(CN)
6
]2±18±2
FAD 4 ± 1 6 ± 2
Duraquinone 1 ± 1 5 ± 1
CuCl
2
2±1 2±1
Cytochrome c ND ND
1,4-Antraquinone ND –
Coenzyme Q
10
ND –
NADP
b
–ND
NADPH
b
–ND

a
A rate of 100% corresponds to 840 mU mg
)1
protein.
b
From
Pedras et al. [31].
Table 5. Effect of the phytoalexins camalexin and cyclobrassinin,
the brassinin regioisomer isobrassinin and fungicide thiabendazole
on BO activity (a complete list with 78 tested compounds is pro-
vided in the supplementary Table S1). BO activity was measured
under standard conditions described in the Experimental proce-
dures; inhibition is expressed as percentage of control activity;
results are expressed as the means ± SD of at least four indepen-
dent experiments.
Compound
Inhibition (%)
0.10 m
M 0.30 mM
Camalexin 30 ± 4 53 ± 4
Cyclobrassinin 23 ± 6 37 ± 8
Thiabendazole 16 ± 3 25 ± 7
Isobrassinin 11 ± 5 23 ± 6
M. S. C. Pedras et al. Brassinin oxidase, a fungal detoxifying enzyme
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3695
investigate the kinetic mechanism of BO. Varying the
concentration of brassinin (0.05–0.30 mm) and keeping
the concentration of PMS constant (0.10, 0.20 and
0.60 lm) gave an intersecting pattern to the left of the
1 ⁄ S axis (Fig. 5A). A second set of experiments was

performed varying the concentration of PMS (0.05–
0.30 lm) and keeping the concentration of brassinin
constant (0.05, 0.10 and 0.15 mm) (Fig. 5B). The inter-
section point was on the 1 ⁄ V axis. Both sets of data
were indicative of a sequential mechanism but did not
distinguish between an ordered or random sequential
mechanism. These two types of kinetic mechanisms
could be distinguished using camalexin as the dead-end
inhibitor of BO. Thus, kinetic data obtained from
experiments performed with various PMS concen-
trations (0.05–0.40 lm) and constant concentrations of
camalexin (0.10 and 0.30 mm) gave the characteristic
plot of uncompetitive inhibition (Fig. 5C). By contrast,
data obtained by varying the concentration of
brassinin (0.05–0.30 mm) and keeping the concentra-
tion of camalexin constant showed that camalexin was
Fig. 4. Lineweaver–Burk plots of BO activities in the presence of
the phytoalexins (A) camalexin and (B) cyclobrassinin. Purified
enzyme obtained from Q-Sepharose chromatography was used for
BO activity measurements. Enzyme activity was determined as
described in the Experimental procedures.
Fig. 5. Distinguishing ping-pong versus sequential kinetic mecha-
nisms for BO. (A) Lineweaver–Burk plot for the oxidation of brassi-
nin carried out in the presence of a fixed concentration of PMS and
varied [brassinin]. (B) Lineweaver–Burk plot for the oxidation
of brassinin carried out in the presence of a fixed concentration of
brassinin and varied [PMS]. (C) Distinguishing ordered sequential
versus random sequential mechanisms for BO. Lineweaver–Burk
plot for the dead-end inhibition of BO by camalexin at the indicated
concentrations of PMS in the presence of a fixed concentration of

brassinin at 0.60 m
M.
Brassinin oxidase, a fungal detoxifying enzyme M. S. C. Pedras et al.
3696 FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS
a competitive inhibitor (Fig 4A). These results demon-
strate that BO catalysis occurs through an ordered
mechanism in which brassinin binds first to the enzyme
followed by PMS binding to the BO binary complex.
Analysis of BO activity in plants inoculated with
L. maculans
B. napus plants susceptible to infection by L. maculans
and Brassica juncea plants resistant to infection by
L. maculans BJ 125 were inoculated, incubated and
analyzed for BO activity. The results obtained
(Table 6) demonstrate that only infected leaves and
stems of the susceptible plants exhibited BO activity;
no BO activity was found in non-inoculated stems or
leaves or inoculated resistant plants (Table 6). Further-
more, analysis of phytoalexin production showed the
presence of methoxybrassinin and spirobrassinin in
infected leaves of B. napus [5].
Mycelia extracts of cultures of L. maculans BJ 125
showed BO activity when cultures were induced with
3-phenylindole but only traces in control cultures.
These analyses confirm that BO activity in L. maculans
is inducible.
Discussion
The present study reports the purification and charac-
terization of BO, a phytoalexin detoxifying enzyme
produced by the plant pathogenic fungus L. maculans

both in infected plants and in axenic fungal cultures.
This enzyme is a monomer with an apparent molecular
mass of 57 kDa that catalyzes the transformation of
the dithiocarbamate toxophore of brassinin into the
corresponding nontoxic aldehyde (Fig. 1). BO appears
to be the first enzyme that has been described to cata-
lyze this unique functional group transformation. A
peak of BO activity obtained by chromatofocusing was
observed at pH 7.1–7.2, suggesting this to be the pI of
the enzyme.
Elution of BO from a concanavalin A Sepharose
column suggested it to be glycosylated [23]. Concanav-
alin A affinity chromatography has been used for puri-
fication of secreted proteins N-glycosylated with sugars
such as d-glucose and d-mannose [18,24,25]. To dem-
onstrate that BO was indeed a glycosylated protein,
purified BO was deglycosylated using either PNGase F
or endo-b-N-acetylglucosaminidase and the molecular
mass of the native and deglycosylated forms of enzyme
were compared by SDS ⁄ PAGE. Treatment of BO with
either N-glycosidase caused a decrease in the apparent
molecular mass of BO of approximately 20% (Fig. 3).
PNGase F and endo-b-N-acetylglucosaminidase are
enzymes used for the release of N-linked glycans from
glycoproteins [26,27].
Taken together, the assays used for cellular locali-
zation (Table 2) and the glycosylation analysis (Fig. 3)
of BO suggest that this enzyme is localized in the cell
wall. This cellular localization of BO could allow a
more efficient detoxification of brassinin. In this con-

text, it is pertinent to point out that the enzyme cata-
lyzing the detoxification of the phytoalexin kievitone,
kievitone hydratase (EC 4.2.1.95), is also a glyco-
enzyme secreted by the bean fungal pathogen Fusarium
solani f. sp. phaseoli [28].
The peptides deduced from the LC-ESI-MS ⁄ MS
spectral data of purified BO digested with trypsin
(Table 3) did not show a significant match with other
proteins available in the NCBI blast database. Anal-
yses of these peptides using the NCBI blast database
pertaining to fungi showed that some peptides in
Table 3 had homology with different putative oxido-
reductases (results not shown). In addition, the
majority of peptides in Table 3 showed some homo-
logy to a putative short-chain dehydrogenase from
A. terreus NIH2624 and putative NADP-dependent
flavin oxidoreductase from A. nidulans FGSC A4.
These peptide sequences (Table 3) should be sufficient
for identification of the complete sequence of the
enzyme when the genome sequence of L. maculans is
available [sequencing of the genome of L. maculans
is in progress ( />English/Projets/#region)].
Table 6. BO activity in plants infected with L. maculans isolate BJ
125. Tissues of B. napus cv. Westar (susceptible) and B. juncea cv.
Cutlass (resistant) were homogenized in buffer and protein extracts
were assayed for BO activity, as described in the Experimental
procedures. BO activity was determined in protein extracts of
mycelia of L. maculans isolate BJ-125 (control cultures and cul-
tures incubated with 3-phenylindole, 0.05 m
M). The results are

expressed as the means ± SD of four independent experiments.
lmolÆmin
)1
= U; ND, not detected.
Tissues analyzed for BO activity
Specific activity
(mUÆmg
)1
)
Control leaves – B. napus ND
Inoculated leaves – B. napus 1.10 ± 0.05
Control stems – B. napus ND
Inoculated stems – B. napus 1.41 ± 0.05
Control leaves of whole plants – B. napus ND
Inoculated leaves of whole plants – B. napus 0.52 ± 0.07
Control leaves – B. juncea ND
Inoculated leaves of B. juncea ND
Control mycelia – L. maculans Traces
a
Mycelia incubated with 3-phenylindole –
L. maculans
2.31 ± 0.15
a
£ 0.01 mUÆmg
)1
.
M. S. C. Pedras et al. Brassinin oxidase, a fungal detoxifying enzyme
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3697
The wide range of cofactors that serve as electron
acceptors of BO (PMS, small quinones or FMN) dem-

onstrate that BO is not selective with respect to elec-
tron acceptors (Table 4). Interestingly, PMS was a
more efficient electron acceptor than some natural
cofactors (e.g. FMN, FAD). Because BO has no cova-
lently attached cofactor, as indicated by UV-visible
spectroscopic analysis, it is possible that natural elec-
tron acceptors of BO could be components of the cell
wall of L. maculans. Some fungi can produce extra-
cellular quinone derivatives used in the biosynthesis of
melanin [29] and other metabolites. For example, the
brown rot fungus Gloeophyllum trabeum secreted two
quinone derivatives used to reduce Fe
3+
and produce
H
2
O
2
[30].
In view of the important role of brassinin in crucifer
phytoalexin biosynthesis and its effective detoxification
by L. maculans, inhibitors of BO are being developed
[7,13]. Toward this end, the effects of the phytoalexins
camalexin, 1-methylcamalexin, cyclobrassinin and
rutalexin, the commercial fungicide thiabendazole, and
several synthetic compounds (see supplementary
Table S1) on BO activity were evaluated. Unexpect-
edly, the phytoalexins camalexin and cyclobrassinin
were the best inhibitors of BO activity, whereas none
of the designed compounds (supplementary Table S1)

showed inhibitory effects. In addition, none of these
compounds (supplementary Table S1) were trans-
formed by BO. An additional surprise was revealed by
kinetic analyses of the inhibition of BO activity
because both camalexin and cyclobrassinin were shown
to be competitive inhibitors (Fig. 4). These molecules
are the first inhibitors reported for a phytoalexin
detoxifying enzyme. In addition, because these inhibi-
tors are also phytoalexins, this discovery indicates that
the various constituents of a phytoalexin blend have
multiple physiological functions. For example, in addi-
tion to antimicrobial activity, constituents of these
blends may inhibit specific enzymes produced by fun-
gal pathogens. Furthermore, it is of interest to note
that L. maculans is able to metabolize and detoxify
cyclobrassinin but unable to metabolize camalexin [31].
Both camalexin and cyclobrassinin are biosynthesized
from l-tryptophan; however, although cyclobrassinin
is derived from brassinin and both co-occur in various
cultivated species [5], camalexin appears to be pro-
duced only in wild species (e.g. Camelina sativa and
Arabidopsis thaliana) and is biosynthesized by a diver-
gent pathway [32]. Furthermore, it should be noted
that camalexin (and the synthetic compound 3-pheny-
lindole) could induce BO production substantially,
whereas the phytoalexin spirobrassinin (and thiabenda-
zole, a commercial fungicide) displayed no apparent
effect. That the induction of BO was not related with
the antifungal activity of these compounds was clari-
fied by thiabendazole, which was a 50-fold more

potent fungicide than camalexin but did not induce
BO [31]. Due to the substantial inhibitory effect of
camalexin on BO activity, a decrease of the rate of
brassinin detoxification in cultures of L. maculans
co-incubated with brassinin and camalexin was
expected. However, our previous results did not show
such a rate decrease [31]. This apparent discrepancy
between the results obtained with cell cultures [31] and
the current results obtained with purified BO could be
due to two opposite effects of camalexin: (a) induction
of BO and (b) inhibition of BO activity. Therefore,
the overall result was no detectable change in brassi-
nin transformation rates in cultures of L. maculans.
Nonetheless, because plants producing camalexin and
brassinin were unknown until now, this apparent con-
tradiction has not been investigated. Without doubt, it
would be most interesting to evaluate the disease resis-
tance of such plants, which may be substantially higher
because camalexin is not detoxified by crucifer patho-
genic fungi such as blackleg or blackspot [7] and is a
potent mycelial growth inhibitor of L. maculans (com-
plete inhibition at 0.5 mm) [31].
Recently, we proposed a mechanism for the trans-
formation of brassinin to indole-3-carboxaldehyde [14],
which invoked the formation of an imido dithiocar-
bamate intermediate (I
1
) partly resembling a cyclo-
brassinin structure, followed by formation of a fully
conjugated intermediate (I

2
) partly resembling a cama-
lexin structure (Fig. 6). Because both cyclobrassinin
and camalexin are competitive inhibitors of BO, these
results lend support to the previously proposed reac-
tion mechanism. On the other hand, the absence of
inhibition observed in the presence of N¢-methylbrassi-
nin and 1-methylcamalexin suggests that these mole-
cules do not fit in the active site of BO. Furthermore,
competitive inhibition is consistent with our steady-
state kinetic studies indicating that BO followed an
ordered kinetic mechanism (using PMS as electron
acceptor and camalexin as dead-end inhibitor; Figs 4
and 5). This characteristic of BO is in contrast with
flavoenzymes [33] and quinoenzymes [34,35] containing
a covalently bound cofactor, which are known to
display a ternary complex or ping-pong kinetic mecha-
nism. Interestingly, plant cytokinin oxidases ⁄
dehydrogenases (CKXs) catalyze the irreversible degra-
dation of cytokinins (secondary amines) to aldehydes
in a single enzymatic step [36]. This oxidative cleavage
of the side chain of cytokinins is somewhat related to
the degradation of brassinin by BO. In addition, some
CKXs appear to be glycosylated and can transfer
Brassinin oxidase, a fungal detoxifying enzyme M. S. C. Pedras et al.
3698 FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS
electrons to artificial electron acceptors such as PMS
and coenzyme Q
0
[37–40], similar to BO. Yet, unlike

BO, CKXs have FAD covalently bound and the cata-
lytic cycle occurs through a ternary complex mecha-
nism [33]. That is, comparison of the characteristics
and function of BO with ‘somewhat similar’ enzymes
emphasizes its uniqueness and explains its lack of
sequence homology to proteins available in current
databanks.
Analysis of BO activity in plant tissues (stem and
leaf) susceptible and resistant to L. maculans, cvs.
Westar (B. napus) and Cutlass (B. juncea), respec-
tively, revealed that BO is produced only in suscepti-
ble plants (Table 6). That is, BO is an enzyme
produced in vivo in susceptible tissues but not in resis-
tant ones, during infection by L. maculans. Further-
more, production of BO in vitro fungal cultures
requires induction with specific compounds (e.g.
3-phenylindole) (Table 6). Taken together, these
results demonstrate that BO is not an inconsequential
enzyme produced just when the pathogen has all
growth requirements satisfied. By contrast, BO is per-
haps one of the best arms used by the pathogen
L. maculans to overcome the inducible antifungal
plant defenses (phytoalexins). In this context, it is
pertinent to recall the precursor function of brassinin
vis-a
`
-vis phytoalexins and thus the negative impact on
the plant if it is depleted of it.
Detoxification of phytoalexins from the family Legu-
minosae has shown the significance of phytoalexin

detoxification in the interaction of plants with fungi
[7]. Pioneering work on the detoxification of the phyto-
alexin pisatin by pisatin demethylase, produced by the
plant pathogenic fungus Nectria haematococca, demon-
strated that this enzyme functioned as a virulence trait
[41]. Such a precedent and our overall results indicate
that BO could be a virulence trait of L. maculans as
well, a product of pathogen evolution over numerous
life cycles of interaction with brassica plants.
The apparent role of BO in the pathogenicity of
L. maculans may be confirmed once the gene(s) for this
enzyme has been cloned. Notwithstanding future dis-
coveries, a first generation of BO inhibitors able to
protect plants from fungal attacks by L. maculans
can now be modeled on the structural elements of
camalexin, a ‘natural inhibitor’. In addition, purified
BO will facilitate in vitro evaluation and optimization
of such inhibitors, which could be developed into
selective crucifer protectants after toxicity screens.
Experimental procedures
General experimental procedures
Chemicals and deglycosylating enzymes were purchased
from Sigma-Aldrich (Oakville, Canada) and chromatogra-
phy media and buffers from GE Healthcare (Quebec, Can-
ada). HPLC analysis was carried out with a system
equipped with a quaternary pump, an automatic injector, a
photodiode array detector (wavelength range 190–600 nm),
a degasser and Hypersil octadecylsilane column (5 micron
particle size silica, 200 · 4.6 mm), and an in-line filter. The
retention times (t

R
) are reported using a linear gradient elu-
tion with CH
3
CN-H
2
O, 25 : 75 to CH
3
CN, 100%, for
35 min at a flow rate of 1.0 mLÆmin
)1
. All operations
regarding protein extraction, purification and assays were
carried out at 4 °C, except where noted otherwise. Solvents
used in syntheses were treated as previously reported [13].
Fungal cultures
Fungal spores of L. maculans virulent isolate BJ 125 were
obtained from the IBCN collection, Agriculture and Agri-
Food Canada Research Station (Saskatoon, Canada).
Cyclobrassinin
Camalexin
Brassinin
Fig. 6. Proposed mechanism of transformation of brassinin to indole-3-carboxaldehyde catalyzed by BO [14]: note the similarity of the chem-
ical structures of the phytoalexins cyclobrassinin and camalexin and those of intermediates I
1
and I
2
, respectively.
M. S. C. Pedras et al. Brassinin oxidase, a fungal detoxifying enzyme
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3699

Liquid cultures were initiated as described previously
[13,42] and induced with 3-phenylindole (0.05 mm) after
48 h. The cultures were incubated for an additional 24 h
and then gravity filtered to separate mycelia from culture
broth.
Preparation of protein extracts
Frozen mycelia (22 g) obtained from cultures of L. macu-
lans (or plant tissues) were suspended in ice-cold extraction
buffer (20 mL) and ground (mortar) for 10 min. The
extraction buffer consisted of 25 mm diethanolamine
(DEA) (pH 8.3), 5% (v ⁄ v) glycerol, 1 mm d,l-dithiothreitol
and 1 : 200 (v ⁄ v) protease inhibitor cocktail (P-8215;
Sigma-Aldrich). The suspension was centrifuged for 60 min
at 58 000 g. The resulting supernatant (20 mL) was used
for chromatographic analyses.
Chromatographic purification of the enzyme
exhibiting BO activity
In step 1, the soluble protein extract from mycelia (20 mL)
was equilibrated by dialyzing against 20 mm Tris–HCl buf-
fer (pH 8.0) containing 2% glycerol (v ⁄ v) and loaded on a
DEAE-Sephacel (Amersham Biosciences, Uppsala, Sweden)
anion-exchange column (1.6 · 12 cm). Proteins were eluted
with the same buffer, first alone and then with a 0.0–0.40 m
NaCl gradient. Fractions (5 mL) were collected and 100 lL
assayed for BO activity. Peak fractions (8–13) showing BO
activity were pooled and used in the second step of purifica-
tion. In step 2, fractions showing BO activity from step 1
(30 mL) were concentrated to 6 mL, equilibrated in 25 mm
ethanolamine buffer (pH 9.4) and applied to a column
(0.9 · 20 cm) of Polybuffer exchanger PBE 94 resin (GE

Healthcare) equilibrated in the same buffer. Elution was
performed with Polybuffer 96, ten-fold diluted with distilled
water and adjusted to pH 6.0. Fractions of 3 mL were col-
lected and 50 lL of each fraction were assayed for BO
activity. A peak of BO activity was observed at pH 7.1–7.2.
In step 3, pooled fractions 38–40 showing BO activity after
step 3 were concentrated to 500 lL and fractionated by fast
protein liquid chromatography (GE Healthcare) on a
Superdex 200 HR10 ⁄ 30 column, pre-calibrated with the fol-
lowing markers of known molecular mass: bleu dextran
(2000 kDa), BSA (67 kDa), ovalbumin (43 kDa), chymo-
trypsin (25 kDa) and ribonuclease (13.7 kDa). Equilibration
and elution were performed at 8 °C with 25 mm Tris-HCl
(pH 8.0), 1% glycerol and 0.15 m NaCl. Fractions of
0.5 mL were collected at a flow rate of 0.4 mLÆmin
)1
, and
10 lL of each fraction were assayed for BO activity. In
step 4, the protein extract of 1.5 mL obtained from step 3
was equilibrated by dialyzing against 20 mm DEA buffer
(pH 8.3) and 1% glycerol. The protein extract was loaded
on a Q-Sepharose (GE Healthcare) cation-exchange column
(1.0 · 5 cm). The proteins were eluted with the same buffer,
first alone and then with a 0.0–0.3 m NaCl discontinuous
gradient using 2.5 mL of NaCl solution, increasing by
0.025 m. Fractions (1 mL) were collected and 50 lL
assayed for BO activity. Peak fractions 14–15 were pooled
and concentrated to 500 lL, and then used for biochemical
analysis.
Analysis of deglycosylated BO

Purified BO was treated with PNGase F (G5166) or endo-
b-N-acetylglucosaminidase (A-0810) following the manu-
facturer’s protocols. Reactions were incubated at 37 °C
overnight with 1 l L (7.7 units) of PNGase F in nondena-
turing and 3 h in denaturing (0.2% SDS, 50 mm b-mercap-
toethanol and 1% of Triton X-100) conditions in the
appropriate buffer (30 lL of total reaction volume). Endo-
b-N-acetylglucosaminidase (1 lL: 5 mU) was incubated
with purified BO at 37 °C for 3 h in denaturing (0.2%
SDS, 50 mm b-mercaptoethanol) conditions with the appro-
priate buffer (30 lL of total reaction volume). After
incubation, 3 lL of SDS ⁄ PAGE buffer was added to each
reaction and samples were analyzed by SDS ⁄ PAGE.
SDS
⁄
PAGE
Protein-denaturing SDS ⁄ PAGE was carried out using 10%
polyacrylamide gels. Standard markers (molecular mass
range 25–200 kDa; Bio-Rad, Hercules, CA, USA) were
used to determine the approximate molecular masses of
purified proteins in gels stained with Coomassie brilliant
blue R-250.
Identification of tryptic peptides of BO by
LC-ESI-MS
⁄
MS
Analyses were carried out by the Plant Biotechnology Insti-
tute, National Research Council of Canada (Saskatoon,
Canada). Protein gel slice was manually excised from Coo-
massie stained gels and placed in a 96-well microtitre plate.

The protein was then automatically destained, reduced with
dithiothreitol, alkylated with iodoacetamide and digested
with porcine trypsin [43] (sequencing grade; Promega, Mad-
ison, WI, USA) and the resulting peptides transferred to a
96-well PCR plate · 3; all steps were performed on a Mass-
PREP protein digest station (Waters ⁄ Micromass, Manches-
ter, UK). The digest was evaporated to dryness, then
dissolved in 20 lL of 1% aqueous TFA, of which 5 lL was
injected onto a NanoAcquity UPLC (Waters, Milford,
MA, USA) interfaced to a Q-Tof Ultima Global hybrid
tandem mass spectrometer fitted with a Z-spray nanoelec-
trospray ion source (Waters ⁄ Micromass). Solvent A con-
sisted of 0.1% formic acid in water, whereas solvent B
consisted of 0.1% formic acid in acetonitrile. The peptide
digest sample was loaded onto a C18 trapping column
Brassinin oxidase, a fungal detoxifying enzyme M. S. C. Pedras et al.
3700 FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS
(Symmetry 180 lm · 20 mm; Waters) and washed for
3 min using solvent A at a flow rate of 15 lLÆmin
)1
. The
trapped peptides (C18 trapping column) were eluted onto a
C18 analytical column (1.7 lm BEH130 C18
100 lm · 100 mm; Waters). Separations were performed
using a linear gradient of 95 : 5% to 45 : 55% A : B over
45 min. The composition was then changed to 20 : 80%
A : B and held for 10 min to flush the column before
re-equilibrating for 7 min at 100 : 0% A : B. Mass calibra-
tion of the Q-Tof instrument was performed using a prod-
uct ion spectrum of Glu-fibrinopeptide B acquired over the

m ⁄ z range 50–1900. LC-MS ⁄ MS analysis was carried out
using data dependent acquisition, during which peptide pre-
cursor ions were detected by scanning from m ⁄ z 400–1900
in TOF MS mode. Multiply charged (2+, 3+ or 4+) ions
rising above predetermined threshold intensity were auto-
matically selected for TOF MS ⁄ MS analysis (by directing
these ions into the collision cell where they fragment using
low energy collision induced dissociation by collisions with
argon and varying the collision energy by charge state
recognition); product ion spectra were acquired over the
m ⁄ z range 50–1900. LC-MS ⁄ MS data were processed using
mascot distiller (version 2.1.1.0; Matrix Science, London,
UK; available at ). The main
search parameters were methionine oxidation as differential
modification and trypsin as enzyme. Protein identification
was carried using peptide sequences obtained by automated
interpretation of the MS ⁄ MS by NCBI blast (http://
ca.expasy.org/tools/blast/).
Concanavalin A sepharose chromatography
A 0.5 · 3 cm column was filled with concanavalin A Sepha-
rose (0.5 mL) (Sigma-Aldrich) and washed with 3 mL of
20 mm Tris–HCl, 1 mm CaCl
2
,1mm MnCl
2
, 0.5 m NaCl
buffer (pH 8.0). The soluble protein extract was loaded and
then the column was washed with 10 mL of the buffer. The
column was eluted with 1 m methyl-a-d-glucopyranoside in
the same buffer. Fractions were collected (2 mL per frac-

tion) and 50 l L samples from each fraction were tested for
BO activity.
Protein fractionation for cellular localization
of BO
The protein extract was fractionated by a modified
method of Bridge et al. [44]. The frozen mycelia (15 g)
were shaved over ice-cold extraction buffer (buffer A,
15 mL) and ground (mortar) for 10 min. Buffer A con-
tained 25 mm DEA (pH 8.3), 10% (v ⁄ v) glycerol, 1 mm
dithiothreitol and 1 : 200 (v ⁄ v) protease inhibitor cocktail
(P-8215; Sigma-Aldrich). The grinding juice was centri-
fuged for 20 min at 15 000 g to yield a supernatant and a
pellet (P1). The supernatant was than centrifuged for
60 min at 100 000 g, the resulting supernatant, containing
soluble proteins was assayed for BO activity and the pellet
(P2) was solubilized by treatment with ice-cold extraction
buffer B [6 mL, buffer B: 20 mm DEA (pH 8.3), 5% (v ⁄ v)
glycerol, 1 mm dithiothreitol, 0.015% (w ⁄ v) Triton X-100
and 50 mm NaCl] and ground (mortar) for 5 min. The
suspension was centrifuged at 4 °C for 15 min at 30 000 g
to yield a fraction containing solubilized membrane pro-
teins. The pellet P1 was solubilized by treatment with ice-
cold extraction buffer C [10 mL, buffer C: 20 mm DEA
(pH 8.3), 5% (v ⁄ v) glycerol, 1 mm dithiothreitol, 0.015%
(w ⁄ v) Triton X-100 and 150 mm NaCl] and ground (mor-
tar) for 5 min. The suspension was centrifuged for 15 min
at 30 000 g to yield a fraction containing solubilized cell
wall proteins.
BO activity assays
The reaction mixture contained 20 mm DEA (pH 8.3),

1mm dithiothreitol, 0.1% Triton X-100, 0.60 mm brassinin
(in dimethylsulfoxide, 5 lL), 0.10 mm PMS and 50–100 lL
of protein extract in a total volume of 500 lL. The reaction
was carried out at 24 °C for 20 min. A control reaction
was stopped by the addition of 2 mL of EtOAc at t =0.
The reaction assays were extracted with 2 mL of EtOAc
and concentrated to dryness in a rotary evaporator.
Extracts were dissolved in acetonitrile (200 lL) and
analyzed by HPLC; quantification was carried out using
integration of peak areas of brassinin and indole-3-carbox-
aldehyde and comparison with calibration curves of each
compound [13,14].
pH and temperature profiles
To determine the pH optimum, the temperature was set to
23 °C and the pH varied from 3.0 to 11.0 in 50 mm DEA,
50 mm N-ethylmorpholine and 100 mm morpholine ethane-
sulfonic acid buffer. These three buffers covered the entire
pH range without a significant change in ionic strength.
The determination of temperature dependence was carried
out at pH 8.3 as described above for the BO assay, except
that the temperature was in the range 8–75 °C.
Kinetic analysis
Kinetic parameters of the purified enzyme were determined
for the substrate brassinin in the concentration range 0.05–
1.0 mm. Standard deviation values for assays were < 5%.
Kinetic data were processed using kaleidagraph program
(Synergy Software, Reading, PA, USA) based on Michael-
is–Menten enzyme kinetics [45]. To determine the type of
inhibition, experiments were carried out using as substrate
brassinin in the concentration range 0.10–0.30 mm in the

presence of 0.10 and 0.30 mm of each compound (Table 5;
see also supplementary Table S1).
M. S. C. Pedras et al. Brassinin oxidase, a fungal detoxifying enzyme
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3701
Protein measurements
Protein concentrations were determined by the Bradford
method [46] using the Sigma prepared reagent and BSA as
the standard.
Synthesis and spectroscopic characterization of
phytoalexins and analogue library
Compounds and phytoalexins shown in Tables 5 and sup-
plementary Table S1 were synthesized as previously
reported [13,14], with isobrassinin according to Pedras et al.
[47], and as described below. Spectroscopic (NMR, HRMS,
FTIR, UV) and chromatographic analyses (HPLC, TLC)
of all compounds and phytoalexins indicated a degree of
purity ‡ 98%.
Synthesis of new compounds in supplementary
Table S1 (entry number)
1-Methyl-3-indolylmethylurea (7)
A mixture of N-(3-indolylmethyl)-N-methanamine (100 mg,
0.62 mmol) and KNCO (61 mg, 0.74 mmol) was refluxed
for 60 min. The reaction mixture was cooled to room tem-
perature and the solvent was evaporated. The residue was
suspended into H
2
O (20 mL), was extracted with EtOAc,
and the organic extracts were combined, dried over Na
2
SO

4
and concentrated to dryness. The crude reaction
mixture was subjected to flash column chromatography
(FCC) (silica gel, CH
2
Cl
2
-MeOH, 98 : 2) to afford the title
compound (95 mg, 75%) as a white solid. Melting point:
126–128 °C(CH
2
Cl
2
-MeOH). H PLC t
R
=5.6min.
1
H-NMR
(500 MHz, CD
3
CN): d 7.64 (d, J = 8 Hz, 1 H), 7.36
(d, J = 8 Hz, 1 H), 7.22 (dd, J = 7.5, 7.5 Hz, 1 H), 7.18
(s, 1 H), 7.1 (dd, J = 7.5, 7.5 Hz, 1 H), 5.45 (br s, 1 H,
D
2
O exchangeable), 4.64 (br s, 2 H, D
2
O exchangeable),
4.41 (s, 2 H), 3.76 (s, 3 H).
13

C-NMR (500 MHz, CD
3
CN):
d 158.9, 137.6, 128.0, 127.4, 121.9, 119.4, 119.2, 113.3,
109.8, 35.4, 32.4. FTIR m
max
(KBr): 3379, 3189, 1658, 1607,
1512 cm
)1
. HRMS: m ⁄ z measured 203.1055 (203.1058 cal-
culated for C
11
H
13
N
3
O). EIMS m ⁄ z (% relative abundance)
203 (M
+
, 80), 159 (26), 144 (100), 132 (20).
1-Methyl-3-indolylmethylmethanesulfonamide (20)
Methanesulfonyl chloride (46 lL, 0.60 mmol) was added to
a solution of 1-methyl 3-indolylmethanamine (80 mg,
0.5 mmol) and triethylamine (140 lL, 1.0 mmol) in tetrahy-
drofuran (4 mL) at 0 °C. The reaction mixture was allowed
to stir at room temperature for 60 min, the precipitate
formed was filtered off, the filtrate was concentrated and the
residue was subjected to FCC (silica gel, CH
2
Cl

2
) to give
1-methyl-3-indolylmethylmethanesulfonamide (86 mg, 72%
yield) as a white solid. Melting point: 76–77 °C (CH
2
Cl
2
).
HPLC t
R
= 9.1 min.
1
H-NMR (500 MHz, CD
3
CN): d 7.69
(d, J = 8 Hz, 1 H), 7.39 (d, J = 8 Hz, 1 H), 7.24 (dd,
J = 7, 7.5 Hz, 1 H), 7.19 (s, 1 H), 7.13 (dd, J = 7.5, 7.5 Hz,
1 H), 5.44 (br s, 1 H, D
2
O exchangeable), 4.42 (d, J =6
Hz, 2 H), 2.77 (s, 3 H), 2.83 (s, 3 H).
13
C-NMR (500 MHz,
CD
3
CN): d 137.6, 128.8, 127.3, 122.1, 119.5, 119.2, 110.6,
110.0, 46.8, 39.8, 32.5. FTIR m
max
(KBr): 3292, 1314, 1148,
746 cm

)1
. HREIMS: m ⁄ z measured 238.0784 (238.0776 cal-
culated for C
11
H
14
N
2
O
2
S). EIMS m ⁄ z (% relative abun-
dance) 238 (M
+
, 36), 158 (71), 144 (100), 130 (17), 79 (27).
N-Acetyl-N-methyl-N-(3-indolyl)methanamine (25)
Acetic anhydride (59 lL, 0.58 mmol) was added to a solution
of N-methyl-N-(3-indolylmethyl)amine (86 mg, 0.53 mmol)
and pyridine (19 lL, 0.25 mmol) in CH
2
Cl
2
. After stirring
for 60 min, the reaction mixture was subjected to FCC
(silica gel, CH
2
Cl
2
) to afford 85 mg of N -acetyl-N-methyl-
N-(3-indolyl)methanamine (74% yield) as a brown oil.
HPLC t

R
= 7.2 min.
1
H-NMR (500 MHz, CDCl
3
) mixture
of rotamers (1 : 2): d 8.58 (br s, 1 H, D
2
O exchangeable),
7.74 (d, J = 8 Hz, 1 H), 7.55 (d, J = 8 Hz, 0.5 H), 7.42
(m, 2 H), 7.20 (m, 5 H), 7.06 (s, 1 H), 4.75 (s, 2.5 H), 4.70
(s, 1 H), 3.0 (s, 2 H), 2.92 (s, 3 H), 2.29 (s, 2 H), 2.14 (s,
3 H).
13
C-NMR (500 MHz, CDCl
3
) Mixture of rotamers:
d 171.3, 170.9, 137.0, 136.8, 127.3, 126.5, 124.5, 122.9,
122.7, 122.6, 120.2, 120.2, 119.9, 118.8, 112.4, 112.0, 111.9,
111.6, 47.2, 42.1, 35.4, 33.8, 22.5, 22.0. FTIR m
max
(KBr):
3232, 1625, 744 cm
)1
. HRMS: m ⁄ z measured 202.1107
(202.1106 calculated for C
12
H
14
N

2
O). EIMS m ⁄ z (% rela-
tive abundance) 202 (M
+
, 62), 145 (12), 130 (100) 118 (9).
3-Indolylmethylpropargylamine (26)
A mixture of 3-indolylmethanamine (100 mg, 0.68 mmol),
K
2
CO
3
(94 mg, 0.68 mmol) and propargylbenzenesulfonate
(106 lL, 0.68 mmol) was allowed to stir at room tempera-
ture for 120 min. The reaction mixture was filtered and
subjected to FCC (silica gel, CH
2
Cl
2
) to afford the title
compound (54 mg, 43%) as a colorless oil. HPLC
t
R
= 9.8 min.
1
H-NMR (500 MHz, CDCl
3
): d 8.20 (br s,
1H, D
2
O exchangeable), 7.74 (d, J = 8 Hz, 1 H), 7.38

(d, J = 8 Hz, 1 H), 7.23 (dd, J = 7, 7 Hz, 1 H), 7.16 (m,
2 H), 4.13 (s, 2 H), 3.51 (s, 2 H), 2.19 (s, 1 H).
13
C-NMR
(500 MHz, CDCl
3
): d 136.8, 127.5, 123.4, 122.6, 120.0,
119.3, 114.4, 111.6, 71.9, 53.8, 43.5, 37.7. FTIR m
max
(KBr):
3273, 2846, 886 cm
)1
. HREIMS: m ⁄ z measured 184.1002
(184.1000) calculated for C
12
H
12
N
2
). EIMS m ⁄ z (% rela-
tive abundance) 184 (M
+
, 12), 155 (37), 130 (100).
2-Naphthalenylmethylpropargylamine (49)
A mixture of 2-naphthalenylmethanamine (100 mg,
0.64 mmol), K
2
CO
3
(88 mg, 0.64 mmol) and propargylben-

Brassinin oxidase, a fungal detoxifying enzyme M. S. C. Pedras et al.
3702 FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS
zenesulfonate (100 lL, 0.64 mmol) was allowed to stir at
room temperature for 120 min. The reaction mixture was
filtered and subjected to FCC (silica gel, CH
2
Cl
2
) to afford
the title compound (67 mg, 54%) as a colorless oil. HPLC
t
R
= 10.9 min.
1
H-NMR (500 MHz, CDCl
3
): d 7.83
(m, 4 H), 7.49 (m, 3 H, 4.08 (s, 2 H), 3.49 (s, 2 H), 2.31
(s, 1 H).
13
C-NMR (500 MHz, CDCl
3
): d 137.1, 133.8,
133.2, 128.6, 128.1, 128.0. 127.3, 127.1, 126.3, 126.1, 82.3,
72.1, 52.7, 37.7. FTIR m
max
(KBr): 3273, 806, 751 cm
)1
.
HREIMS: m ⁄ z measured 195.1040 (195.1048 calculated for

C
14
H
13
N). EIMS m ⁄ z (% relative abundance) 195 (M
+
,
34), 142 (100), 115 (15).
1-Naphthalenylmethylpropargylamine (66)
Preparation as reported for 2-naphthalenylmethylpropargyl-
amine substituting 1-naphthalenylmethanamine (100 mg,
0.68 mmol) for 2-naphthalenylmethanamine. The crude
reaction mixture was subjected to FCC (silica gel, CH
2
Cl
2
)
to afford the title compound (58 mg, 46% yield) colorless
oil [48]. Spectroscopic data identical to previously reported
data [48]. HPLC t
R
= 10.7 min.
Methyl N-(a,a-dimethylbenzyl)dithiocarbamate (72)
Carbon disulfide (49.0 lL, 0.81 mmol) was added to a solu-
tion of N,N-dimethylbenzylamine (100 mg, 0.74 mmol) and
triethylamine (197 lL, 1.48 mmol) in pyridine (1 mL) at
0 °C. After stirring for 20 min, MeI (65.0 lL, 0.81 mmol)
was added and the reaction mixture was stirred for an add-
itional 30 min. The reaction mixture was acidified with
H

2
SO
4
(5.0 mL, 1.5 m), was extracted with Et
2
O, the
organic phase was dried (Na
2
SO
4
) and evaporated under
reduced pressure. The crude reaction mixture was subjected
to FCC (silica gel, CH
2
Cl
2
) to afford methyl N-(a,a-dimeth-
ylbenzyl)dithiocarbamate (147 mg, 89% yield) as a white
solid. Melting point: 74–75 °C (CH
2
Cl
2
). HPLC
t
R
= 21.1 min.
1
H-NMR (500 MHz, CD
3
CN): d 8.22 (br s,

1H, D
2
O exchangeable), 7.32 (m, 4 H), 7.21 (br s, 1 H),
2.48 (s, 3 H), 1.80 (s, 6 H).
13
C-NMR (500 MHz, CD
3
CN):
d 197.1, 146.3, 128.4, 126.6, 125.8, 61.5, 29.04, 17.8. FTIR
m
max
(KBr): 1496, 1358, 1000 cm
)1
. HRMS: m ⁄ z measured
225.0644 (225.0646 calculated for C
11
H
15
NS
2
). EIMS m ⁄ z
(% relative abundance) 225 (M
+
, 74), 119 (100).
Methyl N-(a,a-dimethylbenzyl)carbamate (73)
A solution of N,O- bis(trimethylsilyl)acetamide (270 ll,
1.1 mmol) in CH
2
Cl
2

(1 mL) was added to a solution of
N,N-dimethylbenzylamine (100 mg, 0.74 mmol) in CH
2
Cl
2
(6 mL) and the mixture was stirred at room temperature
for 30 min. The reaction was then cooled to 0 °C and a
solution of methyl chloroformate (84 lL, 1.1 mmol) in
CH
2
Cl
2
(1.5 mL) was added, the reaction mixture was
allowed to stir at 0 °C for an additional 60 min, was
quenched with H
2
O (10 mL), extracted with EtOAc and the
organic extract was dried (Na
2
SO
4
) and concentrated. The
residue obtained was subjected to FCC (silica gel, CH
2
Cl
2
)
to give methyl N-(a,a-dimethylbenzyl)carbamate (130 mg,
91% yield) as a white solid. Melting point: 56–57 °C
(CH

2
Cl
2
). HPLC t
R
= 12.9 min.
1
H-NMR (500 MHz,
CD
3
CN): d 7.42 (d, J = 7.5 Hz, 2 H), 7.34 (dd, J = 7.5,
7.5 Hz, 2 H), 7.23 (dd, J = 7, 7.5 Hz, 1 H), 6.02 (br s,
1H, D
2
O exchangeable), 3.52 (s, 3 H), 1.60 (s, 6 H).
13
C-NMR (500 MHz, CD
3
CN): d 155.2, 148.0, 128.1,
126.2, 124.9, 54.7, 50.8, 28.9. FTIR m
max
(KBr): 3352, 2978,
1707, 1374 cm
)1
. HRMS: m ⁄ z measured 193.0634 (193.0637
calculated for C
11
H
15
NS

2
). EIMS m ⁄ z (% relative abun-
dance) 193 (M
+
, 84), 119 (100).
Effect of phytoalexins and analogue library
on BO
To determine inhibitors of BO activity, 78 compounds were
tested. Inhibition experiments were carried out using sub-
strate brassinin at 0.10 mm concentration in presence of 0.10
and 0.30 mm (Table 4) or 0.10 mm (see supplementary
Table S1) of each compound (dissolved in dimethylsulfoxide,
5 lL). The reaction was initiated by addition of purified
BO. Control experiments were carried out using
dimethylsulfoxide.
Analysis of BO activity in plants infected with
L. maculans
Leaves of B. napus susceptible cultivar Westar and B. jun-
cea resistant cultivar Cutlass [12] were inoculated with
spores of L. maculans virulent isolate BJ 125 (10
6
ÆmL
)1
).
Plants were incubated at 23 ± 1 °C for 9 days in a plastic
tent to maintain high moisture conditions (> 90%), under
a 16 h photoperiod. Alternatively, leaves and stems were
excised, placed in separate Petri dishes and inoculated simi-
larly with fungal spores. Leaves and stems were incubated
at 23 ± 1 °C for 7 days under continuous light. For analy-

sis of BO, leaves were frozen in liquid nitrogen and imme-
diately extracted with extraction buffer as reported above
for the preparation of protein extracts.
Acknowledgements
We thank V. K. Sarma-Mamillapalle for synthesis of
isobrassinin. Financial support from the Natural
Sciences and Engineering Research Council of Canada
(Discovery Grant to M. S. C. P.), Canada Foundation
for Innovation (Infrastructure Fund to M. S. C. P.),
Canada Research Chairs program (Research Grant to
M. S. C. P.) and the University of Saskatchewan
M. S. C. Pedras et al. Brassinin oxidase, a fungal detoxifying enzyme
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3703
(Teaching Assistantship to M. J.) is gratefully
acknowledged.
References
1 Ferreira RB, Monteiro S, Freitas R, Santos CN, Chen Z,
Batista LM, Duarte J, Borges A, Artur R & Teixeira RA.
(2006) Fungal pathogens: the battle for plant infection.
Crit Rev Plant Sci 25, 505–524.
2 Field B, Jordan F & Osbourn A (2006) First encoun-
ters-deployment of defence-related natural products by
plants. New Phytol 172, 193–207.
3 Maor R & Shirasu K (2005) The arms race continues:
battle strategies between plants and fungal pathogens.
Curr Opin Microbiol 8, 399–404.
4 Bailey JA & Mansfield JW (1982) Phytoalexins. Blackie
& Son, Glasgow, pp. 1–334.
5 Pedras MSC, Zheng QA & Sarma-Mamillapalle VK
(2007) The phytoalexins from Brassicaceae: structure,

biological activity, synthesis and biosynthesis. Nat Prod
Comm 2, 319–330.
6 VanEtten H, Temporini E & Wasmann C (2001) Phyto-
alexin (and phytoanticipin) tolerance as a virulence
trait: why is it not required by all pathogens? Physiol
Mol Plant Pathol 59, 83–93.
7 Pedras MSC & Ahiahonu PWK (2005) Metabolism and
detoxification of phytoalexins and analogs by phyto-
pathogenic fungi. Phytochemistry 66, 391–411.
8 Pedras MSC (2008) The chemical ecology of crucifers
and their fungal pathogens: boosting plant defenses and
inhibiting pathogen invasion. Chem Rec 8, 109–115.
9 Leroux P (2003) Modes of action of agrochemicals
against plant pathogenic organisms. C R Biol 326,
9–21.
10 Lynn A, Collins A, Fuller Z, Hillman K & Ratcliffe B
(2006) Cruciferous vegetables and colo-rectal cancer.
Proc Nutr Soc 65, 135–144.
11 Howlett BJ, Idnurm A & Pedras MSC (2001) Leptos-
phaeria maculans, the causal agent of blackleg disease of
Brassicas. Fungal Genet Biol 33, 1–14.
12 Pedras MSC, Gadagi RS, Jha M & Sarma-Mamillapalle
VK (2007) Detoxification of the phytoalexin brassinin
by isolates of Leptosphaeria maculans pathogenic on
brown mustard involves an inducible hydrolase. Phyto-
chemistry 68, 1572–1578.
13 Pedras MSC & Jha M (2006) Toward the control of
Leptosphaeria maculans: design, syntheses, biological
activity and metabolism of potential detoxification
inhibitors of the crucifer phytoalexin brassinin. Bioorg

Med Chem 14, 4958–4979.
14 Pedras MSC, Jha M, Minic Z & Okeola OG (2007)
Isosteric probes provide structural requirements essen-
tial for detoxification of the phytoalexin brassinin by
the fungal pathogen Leptosphaeria maculans. Bioorg
Med Chem 15, 6054–6061.
15 Jeng HW, Holmes AR & Cannon RD (2005) Charac-
terization of two Candida albicans surface mannoprotein
adhesins that bind immobilized saliva components. Med
Mycol 43, 209–217.
16 Kawano CY, Chellegatti MA, Said S & Fonseca MJ
(1999) Comparative study of intracellular and extracel-
lular pectinases produced by Penicillium frequentans.
Biotechnol Appl Biochem 29, 133–140.
17 Kaji H, Saito H, Yamauchi Y, Shinkawa T, Taoka M,
Hirabayashi J, Kasai K, Takahashi N. & Isobe T
(2003) Lectin affinity capture, isotope-coded tagging
and mass spectrometry to identify N-linked glycopro-
tein. Nat Biotechnol 21, 667–672.
18 Minic Z, Jamet E, Negroni L, Arsene der Garabedian
P, Zivy M & Jouanin L (2007) A sub-proteome of Ara-
bidopsis thaliana mature stems trapped on concanavalin
A is enriched in cell wall glycoside hydrolases. J Exp
Bot 58, 2503–2512.
19 Betenbaugh MJ, Tomiya N, Narang S, Hsu JT & Lee
YC (2004) Biosynthesis of human-type N-glycans in het-
erologous systems. Curr Opin Struct Biol 14, 601–606.
20 Helenius A & Aebi M (2004) Roles of N-linked glycans
in the endoplasmic reticulum. Annu Rev Biochem 73,
1019–1049.

21 Trombetta ES & Parodi AJ (2003) Quality control and
protein folding in the secretory pathway. Annu Rev Cell
Dev Biol 19, 649–676.
22 Jamet E, Albenne C, Boudart G, Irshad M, Canut H &
Pont-Lezica R (2008) Recent advances in plant cell wall
proteomics. Proteomics 21, 893–908.
23 Carlsson J, Janson J-C & Sparrman M (1998) Affinity
chromatography. In Protein Purification, Principles,
High Resolution and Applications (Janson J-C & Ryden
L, eds), pp. 275–329. VCH Publishers Inc, New York,
NY.
24 Wang L, Li F, Sun W, Wu S, Wang X, Zhang L,
Zheng D, Wang J & Gao Y (2006) Concanavalin
A-captured glycoproteins in healthy human urine. Mol
Cell Proteomics 5, 560–562.
25 Czlapinski JL & Bertozzi CR (2006) Synthetic glyco-
biology: exploits in the golgi compartment. Curr Opin
Chem Biol 10, 645–651.
26 Morelle W & Michalski JC (2007) Analysis of protein
glycosylation by mass spectrometry. Nat Protoc 2,
1585–1602.
27 Ghesquie
`
re B, Van Damme J, Martens L, Vandekerckh-
ove J & Gevaert K (2006) Proteome-wide characteriza-
tion of N-glycosylation events by diagonal
chromatography. J Proteome Res 5, 2438–2447.
28 Li D, Chung KR, Smith DA & Schardl CL (1995) The
Fusarium solani gene encoding kievitone hydratase, a
secreted enzyme that catalyzes detoxification of a bean

phytoalexin. Mol Plant Microbe Interact 8, 388–397.
29 Langfelder K, Streibel M, Jahn B, Haase G & Brakhage
AA (2003) Biosynthesis of fungal melanins and their
Brassinin oxidase, a fungal detoxifying enzyme M. S. C. Pedras et al.
3704 FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS
importance for human pathogenic fungi. Fungal Genet
Biol 38, 143–158.
30 Jensen KA Jr, Houtman CJ, Ryan ZC & Hammel KE
(2001) Pathways for extracellular fenton chemistry in
the brown rot Basidiomycete Gloeophyllum trabeum.
Appl Environ Microbiol 67, 2705–2711.
31 Pedras MSC, Jha M & Okeola OG (2005) Camalexin
induces detoxification of the phytoalexin brassinin in
the plant pathogen Leptosphaeria maculans. Phytochem-
istry 66, 2609–2616.
32 Glawischnig E (2007) Camalexin. Phytochemistry 68,
401–406.
33 Mattevi A (2006) To be or not to be an oxidase: chal-
lenging the oxygen reactivity of flavoenzymes. Trends
Biochem Sci 31, 276–283.
34 Schwartz B & Klinman J (2007) Quinone cofactors. In
Handbook of proteins (Cox MM & Phillips GN Jr, eds),
pp. 636–642. John Wiley & Sons, Ltd, Chichester.
35 Anthony C (1996) Quinoprotein-catalysed reactions.
Biochem J 320, 697–711.
36 Schmulling T, Werner T, Riefler M, Krupkova E &
Bartrina y Manns I (2003) Structure and function of
cytokinin oxidase ⁄ dehydrogenase genes of maize, rice,
Arabidopsis and other species. J Plant Res 116, 241–
252.

37 Galuszka P, Frebort I, Sebela M, Sauer P, Jacobsen S
& Pec P (2001) Cytokinin oxidase or dehydrogenase?
Mechanism of cytokinin degradation in cereals. Eur J
Biochem 268, 450–461.
38 Galuszka P, Fre
´
bortova
´
J, Luhova
´
L, Bilyeu KD,
English JT & Fre
´
bort I (2005) Tissue localization of
cytokinin dehydrogenase in maize: possible involvement
of quinone species generated from plant phenolics by
other enzymatic systems in the catalytic reaction. Plant
Cell Physiol 46, 716–728.
39 Fre
´
bortova
´
J, Fraaije MW, Galuszka P, Sebela M, Pec
P, Hrba
´
c J, Nova
´
k O, Bilyeu KD, English JT & Fre
´
-

bort I (2004) Catalytic reaction of cytokinin dehydro-
genase: preference for quinones as electron acceptors.
Biochem J 380, 121–130.
40 Fre
´
bort I, Sebela M, Galuszka P, Werner T, Schmu
¨
lling
T & Pec P (2002) Cytokinin oxidase ⁄ cytokinin dehydro-
genase assay: optimized procedures and applications.
Anal Biochem 306, 1–7.
41 Delserone LM, McCluskey K, Matthews DE & Van-
Etten HD (1999) Pisatin demethylation by fungal
pathogens and non pathogens of pea: association with
pisatin tolerance and virulence.
Physiol Mol Plant
Pathol 55, 317–326.
42 Pedras MSC & Khan AQ (1996) Biotransformation of
the brassica phytoalexin brassicanal A by the blackleg
fungus. J Agric Food Chem 44, 3403–3407.
43 Rosenfeld J, Capdevielle J, Guillemot JG & Ferrara P
(1992) In-gel digestion of proteins for internal sequence
analysis after one- or two-dimensional gel electrophore-
sis. Anal Biochem 203, 173–179.
44 Bridge PD, Kokubun T & Simmonds MJ (2003) Protein
extraction from fungi. In Methods in Molecular Biology,
Vol. 59. Protein Purification Protocols, 2nd edn (Cutler
P, ed.), pp. 37–46. Humana Press, Totowa, NJ.
45 Atkins GL & Nimmo IA (1980) Current trends in
the estimation of Michaelis–Menten parameters. Anal

Biochem 104, 1–9.
46 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein dye-binding. Anal
Biochem 72, 248–254.
47 Pedras MSC, Suchy M & Ahiahonu PWK (2006)
Unprecedented chemical structure and biomimetic syn-
thesis of erucalexin, a phytoalexin from the wild crucifer
Erucastrum gallicum. Org Biomol Chem 4, 691–701.
48 Haga T, Komiyoji T, Okada H & Mitani S (1991) Prep-
aration of thenylnaphthylmethylamine derivatives as
agrochemical fungicides. Kokai Tokkyo Koho Japanese
patent no. JP 03120272 A 19910.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Separation of BO activity by concanavalin A
affinity chromatography.
Table S1. Effect of phytoalexins and synthetic com-
pounds on BO activity.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
M. S. C. Pedras et al. Brassinin oxidase, a fungal detoxifying enzyme
FEBS Journal 275 (2008) 3691–3705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3705