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: s.pedras@usask.ca (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 (http://www.genoscope.cns.fr/externe/ 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 [...]... 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 FEBS Journal 275 (2008) 369 1–3 705 ª 2008 The Authors Journal compilation ª 2008 FEBS 3703 Brassinin oxidase, a fungal detoxifying enzyme (Teaching Assistantship acknowledged... 1–7 Delserone LM, McCluskey K, Matthews DE & VanEtten HD (1999) Pisatin demethylation by fungal Brassinin oxidase, a fungal detoxifying enzyme 42 43 44 45 46 47 48 pathogens and non pathogens of pea: association with pisatin tolerance and virulence Physiol Mol Plant Pathol 55, 31 7–3 26 Pedras MSC & Khan AQ (1996) Biotransformation of the brassica phytoalexin brassicanal A by the blackleg fungus J Agric... Wasmann C (2001) Phytoalexin (and phytoanticipin) tolerance as a virulence trait: why is it not required by all pathogens? Physiol Mol Plant Pathol 59, 8 3–9 3 7 Pedras MSC & Ahiahonu PWK (2005) Metabolism and detoxification of phytoalexins and analogs by phytopathogenic fungi Phytochemistry 66, 39 1–4 11 8 Pedras MSC (2008) The chemical ecology of crucifers and their fungal pathogens: boosting plant defenses... disease of Brassicas Fungal Genet Biol 33, 1–1 4 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 Phytochemistry 68, 157 2–1 578 13 Pedras MSC & Jha M (2006) Toward the control of Leptosphaeria maculans: design, syntheses, biological activity and metabolism... of two Candida albicans surface mannoprotein adhesins that bind immobilized saliva components Med Mycol 43, 20 9–2 17 16 Kawano CY, Chellegatti MA, Said S & Fonseca MJ (1999) Comparative study of intracellular and extracellular pectinases produced by Penicillium frequentans Biotechnol Appl Biochem 29, 13 3–1 40 17 Kaji H, Saito H, Yamauchi Y, Shinkawa T, Taoka M, Hirabayashi J, Kasai K, Takahashi N & Isobe... Pedras et al 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 supplementary Table S1 were synthesized as previously reported [13,14], with isobrassinin according to Pedras et al... UK; available at http://www.matrixscience.co.uk) 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/) Brassinin oxidase, a fungal detoxifying enzyme soluble proteins was assayed for BO activity... mm brassinin (in dimethylsulfoxide, 5 lL), 0.10 mm PMS and 5 0–1 00 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... N-glycosylation events by diagonal chromatography J Proteome Res 5, 243 8–2 447 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, 38 8–3 97 29 Langfelder K, Streibel M, Jahn B, Haase G & Brakhage AA (2003) Biosynthesis of fungal melanins and their FEBS Journal... analyzed by HPLC; quantification was carried out using integration of peak areas of brassinin and indole-3-carboxaldehyde and comparison with calibration curves of each compound [13,14] Concanavalin A sepharose chromatography A 0.5 · 3 cm column was filled with concanavalin A Sepharose (0.5 mL) (Sigma-Aldrich) and washed with 3 mL of 20 mm Tris–HCl, 1 mm CaCl2, 1 mm MnCl2, 0.5 m NaCl buffer (pH 8.0) . 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. 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,. 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