Substrate specificity and inhibition of brassinin hydrolases, 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. 7412 FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS var. 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 obtained 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 H N S SCH 3 N H CHO A BorC 3 1 N H 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. M. S. C. Pedras et al. Brassinin hydrolases FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS 7413 was 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 chromatography, 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 chromatography, 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 obtained under denaturing conditions with 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. Brassinin hydrolases M. S. C. Pedras et al. 7414 FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS at 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 pep- tide R.LSASEAMEASLAATRR obtained from a BHLmL2 digest (Table 3) had 100% identity with a putative amidase from Sinorhizobium medicae WSM419 (accession no. YP_001314042). Similarly, the peptide K.TSVPQTLMVCETVNNIIGR obtained from a BHAb digest showed 100% identity with different puta- tive amidases from fungi such as Aspergillus oryzae RIB40 (accession no. XP_001825134), Aspergillus nidulans FGSC 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 K.TSVPQTLMVCETVNNIIGR from BHAb and 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 ) Crude homogenate b 26.9 1070 39.8 100 1.0 HiTrapDEAE FF 4.16 525 126.2 49 3.1 Hydroxyapatite 1.86 466 250.3 44 6.3 Superdex 75 0.077 37 484.7 3.5 12 Superdex 200 0.019 16 833.7 1.5 21 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 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 ) Crude homogenate b 29.4 561 19.1 100 1 Phenyl Sepharose 2.060 165 80.3 30 4.2 Hydroxyapatite 0.148 76 514.7 14 27 Superdex 200 0.012 8.7 728.0 1.5 38 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. M. S. C. Pedras et al. Brassinin hydrolases FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS 7415 numerous 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 (S 0.5 0.27 ± 0.02 mm, Hill coefficient was 1.6 ± 0.2 for BHLmL2, Fig. 4A; S 0.5 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 Mn 2+ ,Ca 2+ , Co 2+ ,Ni 2+ and Zn 2+ , 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 Mr 1 2 3 4 5 220 kDa 120 kDa 60 kDa 58 kDa kDa Mr 1 2 3 4 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). Brassinin hydrolases M. S. C. Pedras et al. 7416 FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS was 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, 1mm 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 pK a of 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 5–56 °C; the apparent optimum temperatures of BHLmL2 were 25–30 °C 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). Observed Mr (expt) Mr (calc) Delta Score Peptide 400.9585 1199.8536 1199.5604 0.2932 20 SNYASIMTSAR 401.0218 1200.0435 1199.5856 0.4579 15 LYGSSGISSMAK 430.2589 858.5032 858.5287 )0.0255 25 VISGLSKR 537.2736 1072.5326 1072.5513 )0.0186 29 ELATQAGDLR 584.8255 1167.6364 1167.6798 )0.0434 42 MVPPLTINKR 608.8271 1215.6397 1215.6897 )0.0500 32 VIEMLIQDK 645.3196 1288.6246 1288.7099 )0.0853 33 NRSTVKEGSALK 667.3418 1332.6691 1332.7513 )0.0822 51 FLLEATKERAR 832.4039 1662.7932 1662.8359 )0.0427 40 R.LSASEAMEASLAATRR 844.4123 1686.8100 1686.8974 )0.0874 18 NLSLAELSLLAEQMR 846.4120 1690.8095 1690.8559 )0.0464 28 QSKTAATMAAEAAELAK 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). Observed Mr (expt) Mr (calc) Delta Score Peptide 400.7445 799.4744 799.4552 0.0192 28 GTIGLSPR 416.8871 1247.6395 1247.6986 )0.0591 19 YEALKARLER 428.7643 855.5139 855.4814 0.0326 35 IAAELSPR 443.5553 1327.6440 1327.6844 )0.0404 28 AERASIDGLGPSR 480.7742 959.5338 959.5287 0.0051 30 LDAGVASSLK 550.7971 1099.5797 1099.6032 )0.0235 65 RRPNMAAIR.R 567.2875 1132.5604 1132.6604 )0.1000 47 KLFGLESALR 658.8793 1315.7440 1315.6844 0.0596 42 DATGDKGLRIDR 716.7034 2147.0883 2147.0715 0.0168 32 K.TSVPQTLMVCETVNNIIGR 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%) BHLmL2 BHAb WRK (10 m M) Asp, Glu 95 ± 5 93 ± 4 EDC (10 m M) Asp, Glu 133 ± 8 115 ± 5 IAA (5 m M) Cys 100 ± 3 95 ± 3 PMSF (5 m M) Ser 55 ± 2 51 ± 2 M. S. C. Pedras et al. Brassinin hydrolases FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS 7417 (Fig. 6A) and of BHAb were 20–27 °C (Fig. 6B). The activation energies of BHs were calculated using the Arrhenius equation after determining enzyme activities at 5, 10, 15 and 22 °C. The activation energies were 12 and 13 kJÆmol )1 for 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 V max (nmolÆmin )1 ) S 0.5 (mM) n H 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. Brassinin hydrolases M. S. C. Pedras et al. 7418 FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS Substrate 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 CH 2 ) 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 CH 2 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)– SCH 3 ) of brassinin and 1-methylbrassinin, but none of the BHs exhibited activity towards the brassinin ana- logue methylated at the side-chain (–CH 3 N–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 Brassinin (1) 100 100 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 )1 and for BHAb is equivalent to 0.10 nmolÆmin )1 ). The results are expressed as means and standard deviations of four independent experiments. M. S. C. Pedras et al. Brassinin hydrolases FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS 7419 SCH 3 , dithiocarbamate compound 6) or isosteric groups (e.g. thiolcarbamate and carbamate) except for the dithiocarbonate 16 (–O–C(=S)–SCH 3 ). 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 (CH 3 SH) 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 (CH 3 SH), 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 K i values were deter- N H H N S SCH 3 3 1 Active site N H H N S SCH 3 O Active site O N H NH 2 S H 3 CS + Active site OH +H 2 O C O S 1st tetrahedral intermediate H Ser :B Ser HB O Active site Ser :B :B Ser S H 3 CS O Active site Ser :B O CH 3 SH 2nd tetrahedral intermediate H H + + Fig. 7. Proposed chemical mechanism of catalytic hydrolysis of brassinin (1)by BHLmL2 and BHAb. Brassinin hydrolases M. S. C. Pedras et al. 7420 FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS mined 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 SCH 3 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. M. S. C. Pedras et al. Brassinin hydrolases FEBS Journal 276 (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS 7421 [...]... mm) and dithiothreitol (0.1 mm) on the enzyme activity, using brassinin as the substrate, were investigated strate in the presence of inhibitor (0.10, 0.20 and 0.40 mm) The kinetics of inhibition was transformed into Lineweaver–Burk double reciprocal plots (1 ⁄ S versus 1 ⁄ V) To determine the Ki, the secondary plot was constructed using the apparent S0.5 values obtained in the presence and absence of. .. HPLC using both methods A and B; quantification was carried out using integration of peak areas of 3-indolylmethanamine and a calibration curve of indolyl-3-methanamine (3) Kinetic analysis and protein measurements Kinetic parameters of the purified enzyme were determined for the substrate brassinin (1) in a concentration range of 0.05–1.0 mm The standard deviation values for the assays were < 3% Kinetic... (0.20 mm), and the cultures were incubated for an additional 24 h and then gravity filtered to separate the mycelia from the broth For analysis of BH induction, 72-h-old liquid cultures (20 mL) were co-incubated with camalexin or 3-phenylindole (the final concentrations in culture were 0.012, 0.025, 0.050, 0.125 and 0.250 mm) and after further incubation for 24 h, the mycelia were separated from the culture... M.S.C.P.) and the University of Saskatchewan (Teaching Assistantship to V.K.S.M.) is gratefully acknowledged We thank M Jha for the synthesis of compounds 4, 6, 8, 12–15, 18 and 20 We acknowledge the technical assistance of P B Chumala, Department of Chemistry, for development of the HPLC method B used for amine detection and K Thoms, Department of Chemistry, for GC ⁄ HRMS method development and data... phytoalexin -detoxifying enzyme from the plant pathogen Sclerotinia sclerotiorum Fungal Genet Biol 46, 201–209 29 Pedras MSC, Minic Z & Sarma-Mamillapalle VK (2009) Synthetic inhibitors of the fungal detoxifying enzyme brassinin oxidase based on the phytoalexin camalexin scaffold J Agric Food Chem 57, 2429–2435 30 Pedras MSC & Hossain M (2007) Design, synthesis, and evaluation of potential inhibitors of brassinin. .. analysis of the headspace volatiles of enzymatic reactions, samples were prepared as follows: 1 mL of the reaction mixture in a vial tightly closed with a screw cap and silicon septum containing brassinin (1.0 mm in 5 lL of dimethylsulfoxide) and 100 lL of protein extract was incubated at 23 °C for 24 h Headspace volatiles were withdrawn from the vial using a gas-tight syringe and injected into the GC-MS.. .Brassinin hydrolases M S C Pedras et al instance, mechanism-based inhibitors of the amidasesignature enzyme FAAH, and of other pharmaceutically important drugs, have been successfully developed [31–33] In conclusion, taken together, the current findings suggest that inhibitors of brassinin detoxifying enzymes, such as BHs, can be designed using cyclobrassinin as the lead structure... data acquisition Substrate specificity The substrate specificity of BHs was determined using the synthetic compounds shown in Table 8 (1, 4, 6, 8, 10–16, 18, 20) Stock solutions (100 mm) of each compound were prepared in dimethylsulfoxide BH activity was assayed by mixing 0.1 lg of the purified enzyme with 1 mm of substrate under experimental conditions identical to those described for the BH activity... (2009) 7412–7428 ª 2009 The Authors Journal compilation ª 2009 FEBS 7425 Brassinin hydrolases M S C Pedras et al of the enzyme with metal ions (Mn2+, Ca2+, Co2+, Ni2+ and Zn2+, all used as a chloride salt, 1 mm) for 5 min The reaction was started by the addition of brassinin (1 mm), and the reaction mixtures were then incubated for an additional 40 min at 23 °C In addition, the effects of EDTA (1 mm), 2-mercaptoethanol... BH activity was carried out at pH 8.3, as described above for the BH assay, except that the temperature ranged from 5 to 56 °C The activation energy (Ea) was determined from the slope ()Ea ⁄ R) of Arrhenius plots of log V (nmol of product per minute) versus 1 ⁄ T Financial support from the Natural Sciences and Engineering Research Council of Canada (Discovery Grant to M.S.C.P.), Canada Foundation for . Substrate specificity and inhibition of brassinin hydrolases, detoxifying enzymes from the plant pathogens Leptosphaeria maculans and Alternaria brassicicola M Effect of camalexin and 3-phenylindole on the activity of BH from mycelial cultures of Leptosphaeria maculans (A) and Alternaria brassicicola (B). The results