brassicicola BHAb were accomplished: native BHLmL2 was found to be a tetrameric protein with a molecular mass of 220 kDa, whereas native BHAb was found to be a dimeric protein of 120 kDa
Trang 1hydrolases, detoxifying enzymes from the plant pathogens Leptosphaeria maculans and Alternaria brassicicola
M Soledade C Pedras, Zoran Minic and Vijay K Sarma-Mamillapalle
Department of Chemistry, University of Saskatchewan, Saskatoon, Canada
Introduction
Crucifers (family Brassicaceae, syn Cruciferae) include
a wide variety of crops cultivated around the world,
including the oilseeds rapeseed and canola
(Bras-sica napus and Brassica rapa) and vegetables such ase cabbage (Brassica oleraceae var capitata), cauliflower (B oleraceae var botrytis) and broccoli (B oleraceae
Keywords
brassinin; cyclobrassinin; detoxification;
dithiocarbamate hydrolase; phytoalexin
Correspondence
M S C Pedras, Department of Chemistry,
University of Saskatchewan, 110 Science
Place, Saskatoon, SK, Canada S7N 5C9
Fax: +1 306 966 4730
Tel: +1 306 966 4772
E-mail: s.pedras@usask.ca
(Received 6 May 2009, revised
26 September 2009, accepted 22 October
2009)
doi:10.1111/j.1742-4658.2009.07457.x
Blackleg (Leptosphaeria maculans and Leptosphaeria biglobosa) and black spot (Alternaria brassicicola) fungi are devastating plant pathogens known
to detoxify the plant defence metabolite, brassinin The significant roles of brassinin as a crucifer phytoalexin and as a biosynthetic precursor of sev-eral other plant defences make it important in plant fitness Brassinin detoxifying enzymes produced by L maculans and A brassicicola catalyse the detoxification of brassinin by hydrolysis of its dithiocarbamate group
to indolyl-3-methanamine The purification and characterization of brassi-nin hydrolases produced by L maculans (BHLmL2) and A brassicicola (BHAb) were accomplished: native BHLmL2 was found to be a tetrameric protein with a molecular mass of 220 kDa, whereas native BHAb was found to be a dimeric protein of 120 kDa Protein characterization using LC-MS⁄ MS and sequence alignment analyses suggested that both enzymes belong to the family of amidases with the catalytic Ser⁄ Ser ⁄ Lys triad Fur-thermore, chemical modification of BHLmL2 and BHAb with selective reagents suggested that the amino acid serine was involved in the catalytic activity of both enzymes The overall results indicated that BHs have new substrate specificities with a new catalytic activity that can be designated as dithiocarbamate hydrolase Investigation of the effect of various phytoal-exins on the activities of BHLmL2 and BHAb indicated that cyclobrassinin was a competitive inhibitor of both enzymes On the basis of pH depen-dence, sequence analyses, chemical modifications of amino acid residues and identification of headspace volatiles, a chemical mechanism for hydro-lysis of the dithiocarbamate group of brassinin catalysed by BHLmL2 and BHAb is proposed The current information should facilitate the design of specific synthetic inhibitors of these enzymes for plant treatments against blackleg and black spot fungal infections
Abbreviations
BGT, brassinin glucosyl transferase; BH, brassinin hydrolase; BHAb, brassinin hydrolase from A brassicicola; BHLmL2, brassinin hydrolase from L maculans; BO, brassinin oxidase; HRMS, high-resolution mass spectrometry; L2 ⁄ M2, Laird 2 and Mayfair 2; LC-ESI-MS ⁄ MS, liquid chromatography-electrospray-tandem mass spectrometry.
Trang 2var italica) [1] Cruciferous oilseeds are the third
larg-est source of edible oil, after oil palm (Elaeis
guineen-sis) and soya bean (Glycine max) Both wild and
cultivated crucifers are known to have positive impact
on human health; a high intake of crucifers has been
convincingly associated with a reduced risk of cancer
[2–5] The phytoalexin brassinin (1) is produced by
crucifers, including economically significant oilseed
crops within the genus Brassica [6,7] Phytoalexins are
inducible secondary metabolites with antimicrobial
activity and are produced de novo by plants in
response to stress, including pathogen attack [8,9]
Depending on the type of stress, crucifers biosynthesize
different blends of phytoalexins that appear to have
multiple roles, including microbial growth inhibition
and inhibition of certain fungal enzymes [7,10] The
antifungal activity of brassinin (1) is partly a result of
its dithiocarbamate group, known to be a potent
toxophore present in synthetic agrochemicals used to
control fungi and weeds [11]
It has been shown that some economically important
fungal plant pathogens can detoxify brassinin (1), a
pro-cess that facilitates the microbial colonization of plants
[12] Such a depletion of brassinin (1) in plant tissues is
an ongoing concern because brassinin (1) is a
biosyn-thetic precursor of several other phytoalexins Hence, a
decrease in the concentration of brassinin can make
plants more vulnerable to pathogen attack, while higher
concentrations of brassinin and derived phytoalexins
are expected to contribute to higher plant resistance to
disease Consequently, technologies that prevent
brassi-nin degradation by pathogens could increase the
concentrations of plant defences and decrease the need
to apply fungicides at the onset of disease
Recently, it was shown that a group of isolates of
the phytopathogenic ‘blackleg’ fungus
[Leptosphae-ria maculans (Desm.) Ces et de Not., asexual stage
Phoma lingam (Tode ex Fr.) Desm.], virulent to
canola, detoxified brassinin (1) to
3-indolecarboxalde-hyde (2) using brassinin oxidase (BO) [13] Also,
another group of isolates of L maculans (Laird 2 and
Mayfair 2, hereon called L2⁄ M2), virulent on brown
mustard (Brassica juncea), was shown to detoxify
brassinin (1) via hydrolysis to indolyl-3-methanamine
(3) [14] Assays using cell-free homogenates incubated
with brassinin (1) demonstrated that the putative
hydrolase was induced by brassinin (1),
N¢-methyl-brassinin (a synthetic derivative of compound 1) and
camalexin (a phytoalexin of wild crucifers) Similarly,
the black spot fungus Alternaria brassicicola (Schwein.)
Wiltshire, also a pathogen of crucifers, detoxified
brassinin (1) via hydrolysis to indolyl-3-methanamine
(3) [15] The summary of brassinin detoxification
reac-tions carried out by different fungal phytopathogens is shown in Fig 1
A reasonable approach to control cruciferous phyto-pathogens, such as L maculans and A brassicicola, could utilize plant treatments with designer compounds that we coined paldoxins [6,10] Paldoxins are envi-sioned as nontoxic and environmentally sustainable plant treatments containing a combination of specific synthetic inhibitors of phytoalexin-detoxifying enzymes However, to design paldoxins with such char-acteristics, a significant understanding of the enzymes involved in the detoxification of phytoalexins, includ-ing their substrate specificity as well as the molecular mechanisms of detoxification, is crucial To this end,
we isolated, characterized and determined the substrate specificities of brassinin hydrolase (BH) produced by
L maculans L2 (BHLmL2) and BH produced by
A brassicicola (BHAb) and report hereon the results
of this work
Results and Discussion Induction of BH activity Previous studies have shown that some crucifer phyto-alexins (e.g brassinin and camalexin) [14] and other chemicals (3-phenylindole) [13] could induce the bio-synthesis of phytoalexin-detoxifying enzymes in plant pathogenic fungi Hence, the activity of BH was exam-ined in mycelia obtaexam-ined from cultures of L maculans and A brassicicola incubated with various concentra-tions (0.012–0.25 mm) of camalexin and 3-phenylin-dole After incubation, the cultures were filtered, the mycelia were extracted and centrifuged, and the result-ing cell-free extracts were analyzed for BH activity using brassinin as the substrate (Fig 2) BH activity
N
H S
CHO A
B or C
3
1
N
NH 2
2
Fig 1 Transformation of brassinin (1) to indole-3-carboxaldehyde (2) or to indolyl-3-methanamine (3) in fungal cultures (A) Leptosp-haeria maculans isolates virulent to canola; (B) L maculans isolates virulent to brown mustard; (C) Alternaria brassicicola.
Trang 3was only observed in cell-free extracts of cultures
incu-bated with camalexin or 3-phenylindole
3-Phenylin-dole induced the highest amount of BH activity at the
highest concentration tested in mycelial cultures of
both L maculans and A brassicicola The substantially
higher induction of BH activity caused by
3-phenylin-dole was particularly noticeable in cultures of L
macu-lans (twofold higher than that of camalexin) For this
reason, 3-phenylindole (0.2 mm) was used to induce the biosynthesis of BHs to facilitate their purification
Purification of BH from L maculans and
A brassicicola Enzymes with BH activity were purified from crude pro-tein extracts of mycelia cultures, using brassinin as the substrate, to monitor the enzymatic activity The puri-fied enzymes were designated as BHLmL2 for the BH from L maculans L2⁄ M2, and as BHAb for the BH from A brassicicola Table 1 summarizes the purifica-tion procedure used for BHLmL2 and indicates the degree of purification and yield achieved for each step The purification protocol involved four column chroma-tography separations: anion exchange chromachroma-tography, hydroxyapatite chromatography, gel filtration chroma-tography on Superdex 75 and gel filtration chromato-graphy on Superdex 200 Table 2 summarizes the purification procedure of BHAb and indicates the degree of purification and yield achieved for each step The purification protocol involved three column chro-matography separations: hydrophobic chrochro-matography, hydroxyapatite chromatography and gel filtration chro-matography on Superdex 200 It is important to note that a loss of BH activity obtained from both fungi was observed during purification in the absence of Triton X-100 and glycerol Thus, to prevent enzymatic inacti-vation, Triton X-100 (0.015%) and glycerol (1–3%) were added to all buffers except for the extraction buffer Under these conditions and storage at )20 C, the activities of both BHs were stable for approximately two weeks Fractions with BH activity obtained after the final chromatography step were pooled, concen-trated and used for biochemical analysis
SDS/PAGE of purified BHs The purity of BHLmL2 obtained from L maculans was examined by denaturing SDS⁄ PAGE, which, upon staining with Coomassie Brilliant Blue R-250, revealed two bands with apparent molecular mass values of 58 and 220 kDa (Fig 3A) In addition, Superdex 200 chromatography of the purified BHLmL2 suggested that the native protein was a tetramer because it was eluted at a position corresponding to a molecular mass
of 220 kDa (data not shown), comparable to that
SDS⁄ PAGE Likewise, purified BHAb obtained from
A brassicicola revealed two bands on SDS⁄ PAGE with apparent molecular mass values of 60 kDa and
120 kDa (Fig 3B) Similarly, the purified protein BHAb was eluted from Superdex 200 (data not shown)
Fig 2 Effect of camalexin and 3-phenylindole on the activity of BH
from mycelial cultures of Leptosphaeria maculans (A) and Alternaria
brassicicola (B) The results are expressed as means and standard
deviations of three independent experiments.
Trang 4at a position corresponding to a molecular mass of
about 120 kDa Thus, these data suggest that native
BHAb is a dimer
Identification and chemical modification of the
purified enzymes
To identify BHLmL2 and BHAb, the bands obtained
from SDS⁄ PAGE were digested with trypsin and then
analyzed by LC-MS⁄ MS using mascot algorithms In
total, 11 peptides were deduced from the LC-MS⁄ MS
spectral data (Table 3) for BHLmL2 and 9 peptides
were deduced for BHAb (Table 4) The sequence
simi-larity of the identified peptides was analyzed using the
NCBI blast algorithm Peptide sequences obtained from
BHLmL2 and BHAb were aligned using
SIM-Align-ment Tools This analysis indicated that several peptide
sequences of BHLmL2 and BHAb showed similarity
but none showed 100% identity to each other The
BHLmL2 digest (Table 3) had 100% identity with a
putative amidase from Sinorhizobium medicae WSM419
(accession no YP_001314042) Similarly, the peptide
BHAb digest showed 100% identity with different
puta-tive amidases from fungi such as Aspergillus oryzae
RIB40 (accession no XP_001825134), Aspergillus
nidulansFGSC A4 (accession no XP_682046),
Emeri-cella rugulosa(accession no AAK29061) and Emericella unguis(accession no AAK29062) In addition, analyses
of the complete sequences of these putative fungal amid-ases using Compute pi⁄ mw tool (http://ca.expasy.org/ tools/pi_tool.html) software predicted that their molecu-lar masses were about 60 kDa, which is in agreement with the molecular mass obtained on SDS⁄ PAGE for BHAb Moreover, other peptides (listed in Tables 3 and 4) showed similarity to putative amidases, suggesting once more that BHs from L maculans and A brassici-cola belong to the amidase superfamily Additionally, sequence alignment analyses of fungal putative amidases that have 100% identity with the peptide
malonamidase E2 from Bradyrhizobium japonicum, showed that these amidases have a similar Ser⁄ Ser ⁄ Lys catalytic triad [16–18] (Supplementary Fig S1) Overall, these results suggest that BHs from L maculans and
A brassicicola belong to the amidase-signature super-family of proteins that contain the Ser⁄ Ser ⁄ Lys triad active site
Proteins of the amidase-signature superfamily from various sources exist in multimeric forms, for example, N-acetylmuramyl-l-alanine amidase from Bacillus sub-tilis strain 168 (dimeric form) [19] and N-acetylmura-moyl-l-alanine amidase from human serum [20] Tetrameric forms of amidases were identified for the polyamidase from Nocardia farcinica [21] and for
Table 1 Enzyme yields and purification factors for BHLmL2.
Purification step
Yield
Specific activity (nmol.min)1.mg)1) Recovery (%) a Purification factor (fold) a
Protein (mg) Activity (nmol.min)1)
a
Recoveries are expressed as percentage of initial activity and purification factors are calculated on the basis of specific activities.bMycelia from 1 L cultures yielded approximately 50 mg of protein.
Table 2 Enzyme yields and purification factors for BHAb.
Purification step
Yield
Specific activity (nmol.min)1.mg)1) Recovery (%)a Purification factor (fold)a Protein (mg) Activity (nmolÆmin)1)
a Recoveries are expressed as percentage of initial activity and purification factors are calculated on the basis of specific activities b Mycelia from 1-L cultures yielded approximately 60 mg protein.
Trang 5numerous microbial amidohydrolases that belong to a
family of cyclic amidases [22] In addition, some
enzymes, such as carbamate hydrolases from bacteria,
also exist in multimeric forms [23] Therefore, it is not
surprising to find that purified BH from L maculans is
a tetramer and that from A brassicicola is a dimer
with apparent molecular mass values of 220 and
120 kDa, respectively
To obtain information on the nature of the amino acid residues occurring in the active site of BHs, pro-tein-modifying reagents were used The chemical modi-fication of BH with selective reagents was carried out
by incubating the enzyme with a large excess of reagent The reaction conditions for the modification
of Asp, Glu, Cys and Ser are shown in Table 5 No significant inactivation of BHs from either L maculans
or A brassicicola was observed upon treatment with reagents specific for Asp and Glu [Woodward’s reagent
K and N-(3-dimethylaminopropyl)-N¢-ethylcarbodii-mide] or for Cys (iodoacetamide) However, treatment
of BHs with a reagent specific for Ser (phen-ylmethanesulfonyl fluoride) resulted in 55% and 51% loss of the initial activities of BHLmL2 and BHAb, respectively These results strongly suggest that Ser is involved in the catalytic activity of BHLmL2 and BHAb and are consistent with our sequence analyses indicating their high similarity to amidases containing the Ser⁄ Ser ⁄ Lys catalytic triad The amidase-signature domain is approximately 130 residues in length and includes the conserved motif with the active-site Ser⁄ Ser⁄ Lys residues in which Ser is the nucleophilic residue Amidase-signature enzymes represent a large family of nonclassical serine hydrolases that are wide-spread in nature, exhibit very diverse biological functions and use amides as substrates [16–18,24,25]
Kinetic properties, effects of metal ions, pH optima and temperature
The substrate saturation curves of both BH enzymes were determined in the presence of increasing concen-trations of brassinin (Fig 4), and the corresponding kinetic parameters, calculated on the basis of the Hill equation, are summarized in Table 6 (S0.5 0.27 ± 0.02 mm, Hill coefficient was 1.6 ± 0.2 for BHLmL2, Fig 4A; S0.5 0.24 ± 0.01 mm, Hill coefficient 1.4 ± 0.1, for BHAb, Fig 4B) As shown in Table 6 and Fig 4, the Hill equation provided an excellent fit, with coefficients of determination of 0.992 for BHLmL2 and 0.997 for BHAb In addition, the fits of the
V= f(S) curves were sigmoidal and V⁄ S = f(S) was not a straight line (Fig 4), suggesting kinetic charac-teristics of allosteric enzymes, rather than Michaelis– Menten kinetics Overall, the kinetic parameters of the
BH enzymes were similar and exhibited slightly positive cooperativity for the substrate brassinin The effect of metal ions, such as Mn2+, Ca2+,
Co2+, Ni2+and Zn2+, on the enzyme activity of BHs was examined No changes in enzyme activity were found in the presence of these metal ions for either BHLmL2 or BHAb In addition, no effect of EDTA
kDa
A
B
225
150
100
75
50
35
25
15
225
150
100
75
50
35
25
220 kDa
120 kDa
60 kDa
58 kDa
Fig 3 SDS ⁄ PAGE of fractions and of purified BH enzymes (A)
From L maculans: lane M, marker proteins (molecular mass values
are indicated); lane 1, crude homogenate (40 lg); lane 2,
HiTrap-DEAE-FF pooled fractions (20 lg); lane 3, hydroxyapatite
chroma-tography (20 lg); lane 4, Superdex 75-pooled fractions (2 lg); lane
5, fraction (BHLmL2) after chromatography on Superdex 200
(1.5 lg) (B) From A brassicicola: lane M, marker proteins
(molecu-lar mass values are indicated); lane 1, crude homogenate (30 lg);
lane 2, Phenyl Sepharose pooled fractions (20 lg); lane 3,
hydroxy-apatite chromatography (3 lg); lane 4, fraction (BHAb) after
chromatography on Superdex 200 (1 lg).
Trang 6was found on enzyme activity Thus, these results
sug-gest the absence of divalent metal cations in the active
site of BHs Both enzymes were inhibited by 1.0 mm
of 2-mercaptoethanol and dithiothreitol For example,
1 mm dithiothreitol caused 72% and 85% inhibition of
BHLmL2 and BHAb, respectively, while 1 mm
2-mer-captoethanol caused 85% and 97% inhibition of BHLmL2 and BHAb, respectively
The influence of pH on the activities of the BH enzymes was investigated in the pH range 6–11 The
pH optima were determined to be in the basic range (pH 8.0–10.0) for BHLmL2 and BHAb (Fig 5A,B) Overall, the kinetic properties and pH optimum pro-files of BHLmL2 and BHAb were comparable to those reported for amidases and carbamate hydrolases [23,24,26] In fact, a general base lysine residue is used
in amidases that contain a Ser⁄ Ser ⁄ Lys triad active site such as FAAH [24] The pH rate profiles of FAAH indicated an increase in activity from pH 5–9, reveal-ing a titratable group with a pKaof approximately 7.9, similar to those profiles observed for BHLmL2 and BHAb
The temperature dependence of the activities of the BHs was tested in the range 5–56C; the apparent optimum temperatures of BHLmL2 were 25–30C
Table 3 Masses and scores of tryptic peptides obtained from purified BHLmL2 Observed = mass ⁄ charge of observed peptide; Mr (expt) = observed mass of peptide; Mr (calc) = calculated mass of matched peptide, Delta = difference (error) between the experimental and calculated masses; Score = ions score The peptide shown in bold has 100% identity with a putative amidase from Sinorhizobium medi-cae WSM419 (accession no YP_001314042).
Table 4 Masses and scores of tryptic peptides obtained from purified BHAb Observed = mass⁄ charge of observed peptide; Mr (expt) = observed mass of peptide; Mr (calc) = calculated mass of matched peptide, Delta = difference (error) between the experimental and calculated masses; Score = ions score The peptide shown in bold has 100% identity with putative amidases from Aspergillus oryzae RIB40 (accession no XP_001825134), Aspergillus nidulans FGSC A4 (accession no XP_682046), Emericella rugulosa (accession no AAK29061) and Emericella unguis (accession no AAK29062).
Table 5 Effect of modifying reagents on relative specific activities
of BHs EDC, N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide; IAA,
iodoacetamide; PMSF, phenylmethanesulfonyl fluoride; WRK,
Woodward’s reagent K.
Modifying
reagent
Possible amino acid
residues modified
Specific relative activity (100%)
EDC (10 m M ) Asp, Glu 133 ± 8 115 ± 5
Trang 7(Fig 6A) and of BHAb were 20–27C (Fig 6B) The
activation energies of BHs were calculated using the
Arrhenius equation after determining enzyme activities
at 5, 10, 15 and 22C The activation energies were 12
and 13 kJÆmol)1for BHLmL2 and BHAb, respectively
These results indicated that BHs do not show
substan-tial differences with respect to the apparent optimal
temperatures and the activation energies
Fig 4 Substrate saturation curves of BHs (A) Brassinin saturation
curve for BHLmL2 and (B) brassinin saturation curve for BHAb The
mixture was incubated at 23 C for 45 min in the presence of
increasing concentrations of brassinin (0–1 m M ) The curves
obtained were fitted to the Hill equation using KaleidaGraph Inset,
corresponding Eadie plots.
Table 6 Kinetic parameters of the BHLmL2 and BHAb (kinetic parameters were obtained from the saturation curves presented in Fig 4 fitted to the Hill equation Standard deviation values were obtained from this fit).
Source of BH (brassinin hydrolase)
Kinetic parameters
Vmax(nmolÆmin)1) S0.5(m M ) nH
BHLmL2 Leptosphaeria maculans
0.49 ± 0.02 0.27 ± 0.02 1.6 ± 0.2
BHAb Alternaria brassicicola
0.42 ± 0.01 0.24 ± 0.01 1.4 ± 0.1
Fig 5 pH dependence of BHLmL2 (A) and BHAb (B) activities The enzyme activities were measured using protein extracts obtained after the second purification step.
Trang 8Substrate specificities of purified enzymes
The substrate specificity of the purified enzymes was
tested using various synthetic compounds containing a
dithiocarbamate group or isosteres located at C-3 of
indolyl-3-methyl or naphthyl-1 or 2-methyl moieties
As summarized in Table 7, BHLmL2 and BHAb
showed hydrolytic activity towards brassinin (1),
1-methylbrassinin (4), methyl
tryptaminedithiocarba-mate (8) and methyl tryptopholdithiocarbonate (16)
Brassinin (1) was the best substrate for both BHLmL2
and BHAb; however, BHAb exhibited relatively higher
activity (of about twofold) towards 1-methylbrassinin
(4) than towards BHLmL2 By contrast, the rates of
hydrolysis of methyl tryptopholdithiocarbonate (16) and methyl tryptaminedithiocarbamate (9) catalysed
by BHLmL2 were substantially higher than those catalysed by BHAb In addition, no catalytic activities were observed with thiolcarbamate (10), carbamate (11), urea (12), thiourea (13) or amide (15), indicating that both BHs are functional group specific Moreover, because naphthyldithiocarbamates (18 and 20) were not transformed, it appeared that the indole group is also important for catalysis Finally, the ethyl dithio-carbamate (14) (a homologue of brassinin containing only an additional CH2) was also not transformed, which indicates that the hydrophobic pocket binding the methylthiol group of brassinin is rather small and hence could not accommodate the additional CH2
group It is worthy of note that, similarly to BHLmL2,
L maculans L2⁄ M2 was able to transform methyl tryptaminedithiocarbamate (8) to tryptamine (9) and methyl tryptopholdithiocarbonate (16) to tryptophol (17) Moreover, the rates of transformation of both compounds 8 and 16 in fungal cultures were substan-tially slower than the rates observed for transformation
of brassinin (1) [14] Altogether, these direct correla-tions between BH activities and corresponding biotransformations in fungal cultures appear to suggest that cells of L maculans L2⁄ M2 produce only one type
of dithiocarbamate hydrolase activity Obviously, this information is of great importance to the design of potential paldoxins
Overall, both BHs showed new substrate specifici-ties, because they were only able to hydrolyse the dithiocarbamate functional group (–HN–C(=S)– SCH3) of brassinin and 1-methylbrassinin, but none of the BHs exhibited activity towards the brassinin ana-logue methylated at the side-chain (–CH3N–C(=S)–
Fig 6 Temperature dependence of BHLmL2 (A) and BHAb (B)
activities The enzyme activities were measured using protein
extracts obtained after the second purification step.
Table 7 Relative specific activities of BHs towards the phytoalexin brassinin (1, natural substrate) and synthetic substrates 1-methyl-brassinin (4), methyl tryptaminedithiocarbamate (8) and methyl tryp-topholdithiocarbonate (16).
Substrate ⁄ compound name (number)
Relative specific activity a (%)
of BHLmL2
Relative specific activity a (%)
of BHAb
1-Methylbrassinin (4) 24 ± 2 50 ± 4 Methyl tryptaminedithiocarbamate (8) 15 ± 2 2 ± 1 Methyl tryptopholdithiocarbonate (16) 19 ± 2 5 ± 2
a
Activities are expressed as the percentage of activity compared
to the substrate activity obtained with brassinin (1.0 m M ; 100% of activity for BHLmL2 is equivalent to 0.15 nmolÆmin)1and for BHAb
is equivalent to 0.10 nmolÆmin)1) The results are expressed as means and standard deviations of four independent experiments.
Trang 9SCH3, dithiocarbamate compound 6) or isosteric
groups (e.g thiolcarbamate and carbamate) except for
the dithiocarbonate 16 (–O–C(=S)–SCH3) Amidases,
which act on carbon–nitrogen bonds (EC 3.5.), and
esterases, which act on carbon–oxygen bonds (EC
3.1.), are enzymes with hydrolytic activities similar to
those of BHs, but to the best of our knowledge no
dithiocarbamate hydrolases have been reported to
date BHLmL2 and BHAb are thus the first members
of this new group of enzymes within the amidase
superfamily (EC 3.5)
Identification of the volatile products and
chemical mechanism of BH catalysed reactions
Our previous studies of the biotransformation of
brass-inin (1) in liquid cultures of L maculans (isolates
avir-ulent on canola, now reclassified as L biglobosa [27])
revealed the presence of carbonyl sulphide (COS) and
methanethiol (CH3SH) in headspace volatiles,
suggest-ing that both products originated from
dithiocarba-mate hydrolysis Consequently, it was suspected that,
in addition to amine (3), these volatiles were products
of the enzymatic transformation of brassinin (1) by
BHs To identify these volatile reaction products, BHs
were incubated with brassinin in tightly closed vials, as
described in the Experimental procedures A gas-tight
syringe was used to collect the headspace volatiles in
the vial and to inject them into a GC⁄ high-resolution
mass spectrometry (GC⁄ HRMS) instrument Two
peaks with retention times of 6.0 and 12.5 min were
identified as carbonyl sulphide (O=C=S) and
metha-nethiol (CH3SH), respectively Thus, these analyses
indicated that brassinin was enzymatically transformed
into 3-indolylmethanamine (3), carbonyl sulphide and methanethiol
The chemical mechanism of dithiocarbamate hydro-lysis catalysed by BHs is expected to be similar to the hydrolyses of amides and esters catalysed by amidases and⁄ or esterases, as depicted in Fig 7 First, the sub-strate binds covalently to the active site of hydrolase via the hydroxyl group of Ser, yielding a first tetrahe-dral intermediate stabilized by other amino acid resi-due(s) Next, the free amine is released and a dithiocarbonate–enzyme complex is formed Finally, nucleophilic attack of water on the thiocarbonyl carbon of the enzyme complex forms a second tetrahe-dral intermediate, which then releases the products carbonyl sulphide and methanethiol, and regenerates the free enzyme
Effect of phytoalexins on BH activities
To identify potential inhibitors of BHs, inhibition experiments were carried out using the purified enzyme, brassinin (1) (0.10 mm final concentration) and the phytoalexins brassicanal A, erucalexin,
rutalex-in, brassilexin, camalexin (chemical structures in Fig S2) and cyclobrassinin [7] Interestingly, only cyclobrassinin (Fig 8A) exhibited an inhibitory effect
on both BHs The type of inhibition caused by cyclo-brassinin was determined from the kinetics of inhibi-tion of BHs, shown in the form of Lineweaver–Burk double reciprocal plots (Fig 8B,C) These results showed that the intersection points of all curves for both BHs were on the 1⁄ V axis, strongly suggesting that cyclobrassinin competitively inhibited the BH activities of both enzymes The Ki values were
deter-N
H
S
1
Active site
N H
H
N S
SCH3
O Active site
O
N
NH2
S
H3CS
+
Active site
OH
+H2O
C
O
S
1st tetrahedral intermediate
H Ser :B
Ser HB
O
Active site
Ser :B :B
Ser
S
H3CS
O
Active site
Ser :B
O
CH3SH
2nd tetrahedral intermediate
H H
+
catalytic hydrolysis of brassinin (1) by BHLmL2 and BHAb.
Trang 10mined to be 0.14 ± 0.02 mm for BHLmL2 and
0.41 ± 0.08 mm for BHAb
Previously we found that both phytoalexins
(cyclo-brassinin and camalexin) competitively inhibited
brass-inin oxidase [13] This inhibitory effect was thought to
be a result of the structural resemblance of each phyto-alexin to two different intermediates in the oxidative transformation mediated by brassinin oxidase In this work, the inhibitory effect of cyclobrassinin on the activity of both BHs is probably caused by its struc-tural resemblance to the substrate brassinin (1) Fur-thermore, based on the mechanism proposed for the hydrolysis of brassinin, it was not surprising to find that camalexin had no inhibitory effect
Conclusion and prospects for paldoxin application
In this work, we have purified and characterized BHLmL2 and BHAb, two brassinin detoxifying enzymes that exhibit BH activity BHs are enzymes produced by L maculans and A brassicicola, and which require induction with specific compounds such
as 3-phenylindole and camalexin Importantly, it was demonstrated that both BHs were inhibited by the phytoalexin cyclobrassinin This discovery lends fur-ther support to the hypothesis that phytoalexins have multiple physiological roles in plant protection, which include inhibition of microbial growth and detoxifying enzymes produced by fungal plant pathogens [13] Cyclobrassinin is biosynthetically derived from brassi-nin, and both phytoalexins co-occur in various culti-vated Brassica species [7] To date, two other brassinin detoxifying enzymes have been reported: BO, isolated from L maculans isolates virulent on canola [13]; and brassinin glucosyl transferase (BGT), produced by the fungal phytopathogen Sclerotinia sclerotiorum (SsBGT1) [28] Similarly to BHs, the activities of both
BO and BGT were inducible by 3-phenylindole and camalexin BO was isolated from the wild-type fungus and found to be competitively inhibited by the crucif-erous phytoalexins cyclobrassinin and camalexin Furthermore, recent results have shown that 5-meth-oxycamalexin, a synthetic compound, was the most effective inhibitor of BO [29] Recombinant SsBGT1 was isolated from Saccharomyces cerevisiae after the corresponding gene of S sclerotiorum was cloned The relatively low expression levels of cloned BGT did not allow inhibition studies to be carried out [28]; however, BGT activity in crude cell-free homogenates of S scle-rotiorum was strongly inhibited by 3-phenylindole and 6-fluoro-3-phenylindole [30]
Knowledge that the Ser⁄ Ser ⁄ Lys catalytic triad is probably involved in the catalytic activity of both BHLmL2 and BHAb will greatly facilitate the design
of inhibitors for both enzymes In particular, the devel-opment of mechanism-based inhibitors is anticipated because inactivation of the hydroxyl group of Ser, the probable nucleophile, has ample precedents For
N H S
N SCH3
Cyclobrassinin
A
B
C
Fig 8 Lineweaver–Burk plots of BH activities for (A) BHLmL2 and
(B) BHAb in the presence of the phytoalexin cyclobrassinin.
Enzyme activity was determined as described in the Experimental
procedures.