Tài liệu Báo cáo khoa học: A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio Role of the seventh histidine and accessibility to the active site docx

A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio Role of the seventh histidine and accessibility to the active site ´ Diana Hernandez-Romero1, Antonio Sanchez-Amat1 and Francisco Solano2 Department of Genetics and Microbiology, Department of Biochemistry and Molecular Biology B, University of Murcia, Spain Keywords catechol oxidase; copper enzymes; monophenolase; phenol oxidase; tyrosinase Correspondence F Solano, Department of Biochemistry and Molecular Biology B, School of Medicine, University of Murcia, Murcia 30100, Spain Fax: +34 9683 64150 Tel: +34 9683 67194 E-mail address: psolano@um.es URL: www.um.es/bbmbi (Received 25 July 2005, revised October 2005, accepted 27 October 2005) doi:10.1111/j.1742-4658.2005.05038.x The sequencing of the genome of Ralstonia solanacearum [Salanoubat M, Genin S, Artiguenave F, et al (2002) Nature 415, 497–502] revealed several genes that putatively code for polyphenol oxidases (PPOs) This soil-borne pathogenic bacterium withers a wide range of plants We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type-3 copper ions at the active site Generation of null mutants in those genes by homologous recombination mutagenesis and protein purification allowed us to correlate each gene with its enzymatic activity In contrast with all tyrosinases so far studied, the enzyme NP_518458 shows higher monophenolase than o-diphenolase activity and its initial activity does not depend on the presence of l-dopa cofactor On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o-diphenols and only residual monophenolase activity, behaving as a catechol oxidase These catalytic characteristics are discussed in relation to two other characteristics apart from the six conserved histidines One is the putative presence of a seventh histidine which interacts with the carboxy group on the substrate and controls the preference for carboxylated and decarboxylated substrates The second is the size of the residue isosteric with the aromatic F261 reported in sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site Polyphenol oxidases (PPOs) are a broad group of copper enzymes able to catalyze the oxidation of a great variety of phenols by molecular oxygen [1] Basically, there are two main types of PPO, laccases and tyrosinases, with significant differences at the polypeptidic copper-binding sites [2] and the spectroscopic properties of the metal ions [3,4] Both enzymes are widely distributed in nature The active site of tyrosinases consists of a pair of coupled copper ions called copper type-3 However, blue laccases have up to four copper ions at the active site of three different types, one type-1, one type-2 and a couple of type-3 Tyrosinases catalyse the hydroxylation of monophenols to o-diphenols (cresolase or monophenolase activity) and the subsequent oxidation of o-diphenols to o-quinones (catechol oxidase or diphenolase activity) [5,6] (Fig 1) One of the most common monophenolic substrates in a variety of organisms is tyrosine, justifying the activity tyrosine hydroxylase for monophenolase The product of this hydroxylation is an o-diphenol, dopa, so that the oxidation of this Abbreviations DO, dopa oxidase; dopachrome, 2-carboxy-2,3-dihydroindole-5,6-quinone; PPO, polyphenol oxidase; R3, a wild-type strain of R solanacearum spontaneously resistant to rifampicin; R3-0337–, R3 mutated in RSc0337 gene; R3-1501–, R3 mutated in RSc1501 gene; TH, tyrosine hydroxylase FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 257 A novel tyrosinase from Rastonia solanacearum ´ D Hernandez-Romero et al A B C particular catechol to o-dopaquinone is also called dopa oxidase (DO) activity On the other hand, laccases oxidize mainly p-diphenols and methoxy-substituted monophenols to finally yield, respectively, p-quinones and dimeric quinonic structures (Fig 1) [7] Tyrosinases are responsible for vertebrate cutaneous pigmentation, browning of fruits and vegetables, and morphogenesis and fruiting body formation in fungi All of these processes involve melanin formation In the bacterial kingdom there are some examples of wellcharacterized tyrosinases They were first described in the genus Streptomyces [8,9], but the enzyme has also been reported in other bacteria such as Sinorhizobium meliloti [10] and Marinomonas mediterranea [11] The latter marine bacterium was the first prokaryote described that expresses two different PPOs One of them is a soluble tyrosinase clearly involved in melanin synthesis [11], and the second is a membrane-bound 258 Fig PPO activities These copper enzymes show a wide range of action on phenolic compounds (A) Tyrosinases show two activities, the hydroxylation of monophenols and the oxidation of o-diphenols Different names are used for these activities, as shown in the Figure One of the most common monophenolic substrates is tyrosine, and in that particular case the activities are named tyrosine hydroxylase (TH) and dopa oxidase (DO) (B) In this case, the product of the catalysis, o-dopaquinone is rapidly converted into dopachrome, the colored product measured in the spectrophotometric assay (C) Laccases are different PPOs with the capacity to oxidize p-diphenols or methoxy-monophenols laccase [12,13] with residual tyrosinase activity; its physiological role is uncertain In fact, the role and physiological advantages of the coexistence of several PPOs in the same micro-organism remain unknown The synthesis of melanin in micro-organisms has been related to pathogenesis and virulence [14] Melanization of the infectious cell seems to offer an advantage, as microbial melanin could protect the pathogen against the host cell [15], although melanization in the host cells is also proposed to be part of the defense system against wounding and infection by the pathogen [16] Thus, the timing and location of melanization seems to be essential for the prevalence of one of these two opposing processes In any case, many of the bacteria that express PPO activities are strains that interact with plants such as Rhizobium meliloti [10], Ralstonia solanacearum [17] and the marine epiphyte Microbulbifer degradans [18] R solanacearum has unique and FEBS Journal 273 (2006) 257–270 ª 2005 FEBS ´ D Hernandez-Romero et al relevant features for addressing the molecular determinants of bacterial pathogenicity to plants It is a soil-borne pathogen which naturally infects roots It exhibits a strong and tissue-specific tropism within the host, invading and multiplying in the xylem vessels In addition, this b-proteobacterium has an unusually wide host range The genome of the strain GMI1000 isolated from tomato has been sequenced [19] It contains up to four genes that putatively code for copper PPOs We have recently proved that at least three of these genes are expressed and the corresponding protein products show PPO activity, including two tyrosinase-like enzymes and one laccase [17] The monophenolase activity of tyrosinases is usually coupled to the o-diphenolase activity In fact, it has been proposed that tyrosinase binds monophenols at the active site to directly oxidize these substrates to o-quinones, so that the two activities cannot be separated [20] In spite of this, o-diphenolase activity can be determined by just using o-diphenols as the initial substrate of tyrosinases Tyrosinases show a much higher specific activity for oxidation of o-diphenols (o-diphenolase activity) than for hydroxylation of monophenols (monophenolase or cresolase activity) [5,21] Furthermore, it is quite common in plants to find PPOs that act exclusively as o-diphenolases, with none or a very residual monophenolase activity [16] In animals, as well as a true tyrosinase, there is another protein called Trp1, which can be considered an o-diphenolase because it shows low oxidase activity with two o-diphenols, dopa and 5,6-dihydroxyindole-2-carboxylic acid [22,23] This is the main reason why classical enzymology classifies the same family of proteins with the pair of type-3 copper ions in tyrosinases (monophenol l-dopa-oxygen oxidoreductase, EC 1.14.18.1) and catechol oxidases (o-diphenol–oxygen oxidoreductase, EC 1.10.3.1), but the differentiation between these two types of enzyme is not clear [4] Looking at the sequences of the two enzymes, both show absolute conservation of the histidine residues of the CuA and CuB binding regions and the same Prosite signatures [2,4,6] The low or zero monophenolase ⁄ o-diphenolase ratio is understandable Chemical oxidation of o-diphenols is much easier than hydroxylation of monophenols The noncatalyzed reaction rate for the atmospheric oxygen oxidation of o-diphenols to o-quinones is several orders of magnitude faster than that for monophenol hydroxylation to o-diphenols Pigment cell researchers should be aware that stock solutions of l-dopa darken spontaneously because of its oxidization, especially at neutral or basic pH, but stock l-tyrosine solutions are stable for long periods FEBS Journal 273 (2006) 257–270 ª 2005 FEBS A novel tyrosinase from Rastonia solanacearum In this paper, we show that one of the two tyrosinase-like PPOs produced by R solanacearum displays higher tyrosine hydroxylase (TH) than DO activity To our knowledge, this is the first tyrosinase with this very interesting feature Comparison of the amino acid sequences at the active site with other tyrosinases and catechol oxidases allows us to propose correlations between key residues in the catalytic patterns of these enzymes and whether they act as true tyrosinases (monophenolases plus o-diphenolases) or only o-diphenolases Results Genes encoding putative tyrosinases in R solanacearum After genome sequencing of R solanacearum, two genes that putatively code for tyrosinase-like enzymes were detected by a blast search [19] They were named catechol oxidase (gene RSc0337, protein NP_518458) and tyrosinase (gene RSc1501, NP_519622) When we submitted both sequences to a hierarchical multiple sequence alignment [25], two sets of proteins showing highest sequence similarity were obtained [17] Interestingly, these sets did not overlap The protein NP_518458 was found to be similar to several plant catechol oxidases and a few bacterial proteins (Table 1) Catechol oxidase from sweet potato (Ipomoea batatas) was not in the top five highest scoring proteins, but it is included in the table because it is the only enzyme of this family that has an available crystal structure [4,26] The similarity to plant catechol oxidases supports the initial naming of this protein [19] On the other hand, the proteins with highest sequence similarity to NP_519622 were several Streptomyces tyrosinases (Table 1) This therefore justifies the naming of this enzyme as tyrosinase Mushroom tyrosinase is included in Table because it is the most commonly used tyrosinase in model studies It is important to note that the most characteristic signatures in the sequences are present in both proteins, tyrosinases and catechol oxidases; these include the six histidine residues involved in the binding of a pair of copper ions and other conserved residues [6] However, so far it is not possible to predict from this signature the enzymatic activity that a protein will actually display Isolation of R solanacearum mutants affected in tyrosinase-like activities Strains with mutations in the two genes coding for tyrosinase-like activities were constructed by homo259 Table Alignment of sequences at the CuA and CuB binding sites of PPOs from R solanacearum, NP_518458 (gene RSc0337) and NP_519622 (gene RSc1501) with the proteins showing highest sequence similarity (scores higher than 100 and e values lower than 10)21 in all cases) PPOs from I batata and A bisporus have been included as important model PPOs The six H residues directly involved in copper binding are marked in shadow background, and the other proposed positions related to the monophenolase vs o-diphenolase differences are in bold and higher size Consensus shows the concordance with the conserved positions for tyrosinases [6] A novel tyrosinase from Rastonia solanacearum 260 ´ D Hernandez-Romero et al FEBS Journal 273 (2006) 257–270 ª 2005 FEBS ´ D Hernandez-Romero et al logous recombination Briefly, the gene RSc1501 was amplified by PCR from genomic DNA of a spontaneous RifR R solanacearum wild-type GMI1000 strain which we called R3 The PCR product with a size of  1.6 kb was digested with BamHI to obtain a fragment between the two copper-binding site coding regions and ligated to pBlueScript pKSII(+) with T4 DNA ligase (Invitrogen, San Diego, CA, USA) The ligation mixture was transformed in Escherichia coli DH5a, and transformants selected for ampicillin resistance The plasmid obtained (pBRI15) was digested with EcoRI and SacI, and the internal RSc1501 gene fragment subcloned in the pFSVK plasmid The resulting plasmid (pCN15) was transformed in E coli S17-1 (kpir), and transformants selected for kanamycin resistance The plasmid in this strain was mobilized into spontaneous RifR R3 by conjugation [17] RSc1501 gene disruption in the transconjugants was confirmed by appropriate PCR and product analysis (data not shown) One strain, R3-1501–, was selected for further assays To obtain mutants affected in the RSc0337 gene, an internal fragment of 300 bp between the two copperbinding sites from this gene was amplified using the appropriate forward and reverse primers Then the product was cloned in the pFSVK plasmid using the NcoI and SacI restriction sites The resulting plasmid pCN337 was transformed in E coli and mobilized into R3 as described above for the RSc1501 gene RSc0337 disruption was also confirmed in the transconjugants by PCR [17], and one strain, R3-337–, was selected for further studies A novel tyrosinase from Rastonia solanacearum Fig TH and DO activities in extracts of wild-type R3 R solanacearum and two mutant strains with mutations in the PPO genes RSc0337 and RSc1501 TH activity was determined at pH and 0.05% SDS, and DO activity at pH and 0.02% SDS genes, which was opposite to that expected from the blast homologies and designated names (Fig 2) Moreover, the TH activity in both mutants showed a very different dependence on l-dopa as cofactor to eliminate the characteristic lag period of tyrosinases [8,20,21] Figure shows the rate of TH activity as a function of the concentration of l-dopa cofactor added to the assay mixture R3-1501– extracts have a high TH activity, almost independent of the addition of l-dopa cofactor, and the lag period before reaching the maximal reaction rate without this addition is short ( 40–60 s under standard conditions) The TH activity of R3-0337– extracts is quite low and needs to PPO activity in R solanacearum and mutants affected in genes coding for these proteins R solanacearum showed monophenolase and o-diphenolase activities, represented by TH and DO, respectively The conditions for the PPO enzymatic assays differed with regard to pH and SDS concentration TH activity was higher at pH and 0.05% SDS, but DO showed a sharp peak at 0.02% SDS and pH In fact, the rate of oxidation of l-dopa was much lower at pH 5, but under these conditions the optimal SDS concentration was 0.05%, the same as optimal TH conditions [17] Furthermore, when these activities were determined in cellular extracts of the mutant strains generated and compared with the wild-type strain, we found that each activity was lost in extracts of different mutants Mutation of the RSc0337 gene resulted in loss of almost all TH activity, whereas mutation of the RSc1501 gene resulted in loss of most of the DO activity, indicating a correspondence between both activities and the proteins encoded by the respective mutated FEBS Journal 273 (2006) 257–270 ª 2005 FEBS Fig Dependence of the TH activity of extracts of R3 and the two mutant strains on the presence of cofactor, L-dopa (n) R3; (d) R3-0337– which only expresses the protein NP_519622; (m) R3-1501– which only expresses the protein NP_518458 Lag periods in the absence of L-dopa were, respectively, 58, 286 and 42 s All measurements represent the estimation of net L-dopachrome formed, after subtraction of the blanks in the absence of the substrate, L-tyrosine 261 A novel tyrosinase from Rastonia solanacearum ´ D Hernandez-Romero et al be activated by the addition of l-dopa Its lag period in the absence of l-dopa is  R3 wild-type extracts behave much more like R3-1501– than R30337– This pattern agrees with the presence of two different enzymes with overlapping activities Purification of two enzymes with different affinities for monophenols and o-diphenols Supernatants of bacterial crude extracts obtained from R3 wild-type and mutant strains were submitted to enzyme purification These supernatants, routinely  30 mL, were first concentrated 5–6 times using ultrafiltration membranes (Millipore; cut-off 10 kDa) and applied to CM-Sephadex A-50 chromatography in 0.05 m sodium phosphate buffer, pH 7, according to the basic pI predicted from their amino-acid sequence After elution of unbound proteins, the ionic strength was increased with a salt gradient of NaCl up to 1.5 m to elute proteins bound to the anionic gel Fractions of 1.9 mL were collected, the protein content was monitored (A280), and TH and DO activities were assayed under the respective optimal conditions The purification profiles of bacterial extracts from wild-type (R3-wt), mutant strain R3-1501– affected in the NP_519622 protein and mutant strain R3-0337– affected in the NP_518458 protein are shown in Fig 4, and a summary of the purification is shown at Table Apart from a small amount of DO activity found in the large peak of unbound proteins eluted before application of the salt gradient, two PPOs were eluted in the wild-type strain at high salt concentration,  0.9 and 1.05 m NaCl, respectively The first one had high TH activity, although it also had detectable DO activity under the optimal conditions for this activity (0.02% SDS, pH 7) The second one displayed only DO activity under these conditions Interestingly, the first peak but not the second one was found in the extracts of R3-1501–, and the opposite was observed in extracts of R3-0337– mutant This behavior clearly suggests that these peaks are due to different enzymes, and that the TH activity is due to the NP_518458 protein, whereas the DO activity is mostly due to the NP_519662 protein As these proteins were preliminarily named catechol oxidase and tyrosinase, respectively, this activity profile strongly indicates that the names should be exchanged The main stages of the purification process for the three extracts are summarized in Table The initial total amounts of protein are not the same because we started purification from different amounts of material During the purification process, we obtained 245-fold and 691-fold purification for the two wild-type PPOs, and yields of  30% These purification factors were 262 Fig Purification profiles in CM-Sephadex chromatography of cellular extracts from wild-type and mutant strains After elution of all unretained proteins, a linear gradient of NaCl up to 1.5 M in the same buffer was applied to the column A280, TH and DO stand, respectively, for the profile of UV absorbance (total protein) and enzymatic PPO activities (A) Wild-type R3 strain; (B) R3-1501–; (C) R3-0337– not so high when we used the mutant extracts as starting material The purified peaks of the two PPOs showed purities greater than 90%, as judged by SDS ⁄ PAGE, and apparent molecular masses of the active enzymes of  35 and 50 kDa (Fig 5) The respective specific activities ensure minimum turnover numbers of 750 and 1550 min)1 for the TH activity of the monophenolase and the DO activity of the o-diphenolase, respectively Affinity for carboxylated and decarboxylated phenolic substrates To explore the affinity of the active site of the two PPOs for phenolic substrates and possible correlations between the structural requirements for interaction and FEBS Journal 273 (2006) 257–270 ª 2005 FEBS ´ D Hernandez-Romero et al A novel tyrosinase from Rastonia solanacearum Table Purification of tyrosinase and catechol oxidase (proteins NP_518458 and NP_519622, respectively) from R solanacearum In all cases, TH activity was determined at pH with mM L-Tyr and 0.05% SDS, and DO activity at pH with mM L-Dopa and 0.02% SDS In column A, 49 and are, respectively, the amounts of protein (lg) in the TH and DO activity peaks Yields were calculated with the values in parentheses, which are the three most active fractions from the purification peaks pooled, but maximal purification (n-fold) was calculated from the most active fraction wt, Wild-type Crude Column A: wt, R3 extract (contains both enzymes) Proteins (lg) Total activity of TH (mU) Total activity of DO (mU) Specific activity of TH (mmg)1) Specific activity of DO (mmg)1) Purification (n-fold) ⁄ yield TH (%) Purification (n-fold) ⁄ yield DO (%) Column B: R3-1501– (contains NP_518458) Proteins (lg) Activity of TH (mU) Specific activity of TH (mmg)1) Purification (n-fold) ⁄ yield TH (%) Column C: R3-0337–(contains NP_519622) Proteins (lg) Activity of DO (mU) Specific activity of DO (mmg)1) Purification (n-fold) ⁄ yield DO (%) Ultrafiltrate Purified fraction 79200 3248 3540 41.0 44.7 ⁄ 100 ⁄ 100 48050 1505 1685 31.3 35.1 0.8 ⁄ 46.3 0.8 ⁄ 47.6 49 & (2 peaks) 493 (1097) 278 (869) 10056 30888 245 ⁄ 34 691 ⁄ 25 16000 1997 124.8 ⁄ 100 9200 1140 123.9 ⁄ 57.1 12 210 (579) 17500 122 ⁄ 29 40800 957 23.5 ⁄ 100 10100 605 60 2.5 ⁄ 63 84 (475) 10500 447 ⁄ 50 kDa 90 46 35 20 Fig SDS ⁄ PAGE of the most pure PPO fractions from Fig 3A 1, First peak (elution volume, Ve ¼ 110 mL); 2, second peak (Ve ¼ 123 mL) All the peaks showed purities of at least 90% Similar single bands were obtained with peaks obtained from R3-1501– and R3-0337– Fig Comparison of the monophenolase ⁄ o-diphenolase activities of extracts [m(mg protein))1] from wild-type and mutated R solanacearum strains to carboxylated ⁄ decarboxylated substrates using the L-tyrosine ⁄ tyramine and L-dopa ⁄ dopamine pairs Kinetic parameters are summarized in Table the differences between the two PPOs, the kinetics parameters of carboxylated ⁄ decarboxylated substrates were calculated We used the couples l-tyrosine ⁄ tyramine for the monophenolase activity and l-dopa ⁄ dopamine for the diphenolase activity Standard activities under optimal conditions are shown in Fig 6, and values for Vmax, Km and catalytic efficiencies in Table Concerning monophenolase activity, the enzyme NP_518458 greatly preferred l-tyrosine to tyramine It showed higher Vmax and lower Km for the carboxylated monophenol, which can be more clearly appreciated if the catalytic efficiency (Vmax ⁄ Km) is calculated At pH the affinity for these substrates was slightly lower (data not shown) On the other hand, the enzyme NP_519662 did not show preference for l-tyrosine In fact, this enzyme was a little bit more efficient in tyramine hydroxylation It was almost completely unable to hydroxylate monophenols at pH 5, showing FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 263 ´ D Hernandez-Romero et al A novel tyrosinase from Rastonia solanacearum Table Kinetic parameters for the two PPOs The enzymes were obtained from extracts of R solanacearum strains mutated in the gene encoding the alternative one DaO, Dopamine oxidase; TaH, tyramine hydroxylase Enzyme Activity Vmax (mmg)1) Km (mM) Cat efficiency (mmg.mM)1) NP_518458 TH TaH TH TaH DO DaO DO DaO 254.4 59.2 106.7 198.9 46.8 8.2 1264.0 3075.0 1.32 2.54 0.94 1.18 2.87 0.95 3.53 3.87 192.7 23.3 113.5 168.6 16.3 8.7 358.1 794.6 A NP_519622 NP_518458 NP_519622 a marked loss of affinity for the substrate (the Km increased to  10 mm; data not shown) and low reaction rates Concerning diphenolase activity, the enzyme NP_518458 was a poor catalyst, but again it preferred the carboxylated o-diphenol (l-dopa) over its decarboxylated counterpart, dopamine On the other hand, the NP_519622 protein showed very efficient diphenolase activity, particularly with dopamine Activities with these o-diphenol substrates were higher than 1000 mmg)1 (Table 3), although the affinity was not very high To summarize, protein encoded by RSc0337 is an efficient monophenolase, especially with carboxylated monophenols, but the protein encoded by RSc1501 is an efficient diphenolase, especially with decarboxylated o-diphenols Dopa accumulation in the TH reaction catalysed by the NP_518458 protein Figure 7A shows the stoichiometric formation of 2-carboxy-2,3-dihydroindole-5,6-quinone (l-dopachrome) and l-dopa during the time course of tyrosine hydroxylation The l-dopa accumulated by the spontaneous disproportion of dopaquinone can be titrated at different periods of time by addition of sodium periodate According to the high preference of the enzyme encoded by the RSc0337 gene for the monophenols and the general mechanism for the reaction of tyrosinases (Fig 7B), it can be seen that dopa is not consumed by the enzyme through the o-diphenolase cycle, as it is not a competitor with the monophenolase cycle Stability of PPOs The stabilities of both enzymes, monophenolase NP_518458 and o-diphenolase NP_519662, were stud264 B Fig (A) Time-course accumulation of L-dopachrome and L-dopa during the TH activity of NP_518458 L-dopachrome (n) is formed directly and monitored continuously, but L-dopa (m) was titrated by addition of excess sodium periodate at several fixed times of reaction (B) Catalytic cycles for the monophenolase (up, clockwise) and o-diphenolase (down, anticlockwise) activities MF, Monophenol; DF, o-diphenol; Q, o-quinone; T, tyrosinase T has three different forms during the cycles: met, resting tyrosinase with Cu(II); oxy, oxygenated form with peroxide bound to Cu(II); deoxy, reduced Cu(I) transient form with high affinity for oxygen The efficiency for both cycles depends basically on the affinity of oxyT for the monophenol or o-diphenol The enzymatic product, o-quinone, undergoes a very fast spontaneous disprorportion to regenerated o-diphenol and the ‘chrome’ (see Fig 1B) Dopa can be chemically oxidized very rapidly to dopachrome by sodium periodate ied by heating to 60 °C and exposure to a relatively high concentration (0.5%) of the chaotropic and denaturing agent SDS Note that the concentration is at least 10 times higher than the SDS used for optimal assay conditions (Fig 8) It can be observed that the first PPO is very stable to both treatments, but the second one is labile Discussion We have found two different genes in R solanacearum coding for putative PPO proteins that contain the typical signatures of tyrosinases, including the CuA and CuB binding sites to ligand the copper FEBS Journal 273 (2006) 257–270 ª 2005 FEBS ´ D Hernandez-Romero et al Fig Stability of proteins NP_518458 (TH activity) and NP_519622 (DO activity) in phosphate buffer, pH Both purified PPOs were submitted to heat (60 °C) or high SDS concentration (0.5%) type-3 pair [2,6] In principle, it is unclear what physiological advantages there are for bacteria to express two proteins so similar in terms of enzymatic activity However, this situation has been found previously in other bacteria Genome sequencing of Streptomyces avermitilis also revealed the presence of two tyrosinase-like enzymes, although it was suggested that one of those genes is not expressed, or shows a very low level of transcription [27] In addition, we have reported the existence and expression of a multipotent laccase and a tyrosinase in Marinomonas mediterranea [11,13] We have now found in R solanacearum that the two tyrosinase-like genes and the laccase-like gene are indeed expressed [17] One attractive advantage to having more than one PPO is that these proteins may interact with each other to form a stable and very efficient melanogenic complex It should be taken into account that melanogenesis is related to virulence of the infective micro-organism, but it is also related to defensive roles in the infected cell, so that the place and time of triggering of melanogenesis must be key to the success of one of these two opposite processes In turn, a melanogenic complex has been described in mammals between tyrosinase and tyrosinase related protein [28] The latter can behave as an o-diphenolase-like protein but also as a stabilizing protein for true tyrosinase [29] In R solanacearum, NP_518458 would mainly catalyse the rate-limiting step, monophenol hydroxylation, and NP_519622 would catalyse the second step, oxidation of o-diphenol to o-quinone, or alternatively a stabilization of the former enzyme Studies on possible interactions between the PPOs are underway in our laboratory On the other hand, environmental conditions, for instance acidic or neutral FEBS Journal 273 (2006) 257–270 ª 2005 FEBS A novel tyrosinase from Rastonia solanacearum environmental pH, may also affect the expression of the most appropriate enzyme Apart from the physiological roles and environmental advantages of having several PPOs in the same organism, we have found that the RSc0337 gene codes for an enzyme with high TH activity and lower DO activity, with optimum assay conditions at pH 5, whereas the RSc1501 gene codes for an enzyme that efficiently oxidizes l-dopa, although it also shows low activity with l-tyrosine, as revealed by the residual TH activity detected in the R3-0337– mutant Its optimal activity is at pH These preferred activities of the two PPOs of R solanacearum are opposite to the names assigned to them when the genome of this bacterium was sequenced and the function of these conceptual proteins was proposed [19] On the basis of blast homology, the NP_518458 protein from the RSc0337 gene was named catechol oxidase, and the NP_519622 protein encoded by the gene RSc1501 was named tyrosinase This was logical according to the mathematical algorithm used for the blast search Score and e values depend on several factors, but mostly the total length of the sequence used for the blast The shorter sequence (412 amino acids), coming from the RSc1501 gene, more closely matches the short sequences (Table 1), which are tyrosinases from Streptomyces species [2], and these homologies led to this enzyme being designated a putative tyrosinase The long sequence (496 amino acids), coming from the RSc0337 gene, more closely matches long bacterial tyrosinases and a series of plant catechol oxidases, which are also long This led to the designation of this protein as a putative catechol oxidase It is clear that matching the whole sequence is not a good way of distinguishing tyrosinases from catechol oxidases Having clearly established that protein NP_518458 is a tyrosinase (monophenolase) rather than a catechol oxidase (o-diphenolase), we observed that it is a very unusual tyrosinase as it is a more efficient monophenolase than o-diphenolase and its TH ⁄ DO ratio is clearly higher than In the same way, it does not need l-dopa cofactor to reach maximal tyrosine hydroxylase activity To our knowledge, this feature is not found in any other reported tyrosinase, from Streptomyces to mammals The turnover number of tyrosinases for DO is about 100 times higher than for tyrosine hydroxylation [21] In this regard, fungal and bacterial tyrosinases are very similar, showing a higher kcat and activity with o-diphenols than with monophenols [8] Moreover, the TH ⁄ DO ratio is almost zero in plant catechol oxidases lacking monophenolase activity In general, o-diphenols bind more rapidly to oxy-tyrosinase than monophenols [4,30] However, this tyrosinase from 265 A novel tyrosinase from Rastonia solanacearum Table Monophenolase ⁄ o-diphenolase ratios of PPOs for carboxylated ⁄ decarboxylated substrate pairs DaO, Dopamine oxidase; TaH, tyramine hydroxylase Enzyme Optimum Preference substrate ⁄ pH TH ⁄ DO TaH ⁄ DaO preferred name NP_518458 pH 5.4 7.2 NP_519622 pH 0.08 0.06 Carboxylated monophenols ⁄ tyrosinase Decarboxylated o-diphenols ⁄ catechol oxidase R solanacearum has the opposite kinetic properties In contrast with all other tyrosinases, the TH ⁄ DO data summarized in Table clearly show that the monophenol is the preferred substrate Tyrosinases catalyse monophenolase hydroxylation and ⁄ or o-diphenolase oxidation as shown in Fig 7B Binding of monophenols to resting met-tyrosinase results in the inactive dead-end complex, but binding of o-diphenols leads the enzyme to the oxy-tyrosinase form, the active species for both monophenolase and o-diphenolase activity [5,30–32] About 85% of resting mushroom tyrosinase is found in the met form and 15% in the oxy form, so that the o-diphenol formed by this 15% is enough to recruit the enzyme to the catalytic cycle after a short time, showing the characteristic lag period of tyrosinases before reaching maximal reaction rate [5,7,30,31] Note that the product of the reaction, dopachrome, is chemically formed by a redox disproportion from the true enzymatic product o-quinone (Fig 1) R solanacearum tyrosinase seems to be almost completely in the oxy form, as judged by the absence of lag period in the absence of l-dopa cofactor This indicates that the dead-end inactive complex (Fig 7B) is not formed in this particular enzyme Titration of the amount of l-dopa generated during its TH activity with sodium periodate shows that this o-diphenol is stoichiometrically accumulated with dopachrome (Fig 7A), but this is not so using mushroom tyrosinase (data not shown) These data confirm the great preference of oxy-tyrosinase for monophenols, so that the DO activity is not competing with TH during the course of the reaction, and the chemically generated l-dopa is not consumed The structural difference between catechol oxidases and tyrosinases has not yet been explained Concerning the crucial regions for catalytic activity and substrate affinity, the six copper-binding histidines of the two PPOs not show any differences (Table 1), but some distinctions must exist The most reliable way of exploring this is comparison of crystal structure data 266 ´ D Hernandez-Romero et al The only data so far available are for sweet potato (Ipomoea batata) catechol oxidase [26] The catalytic copper center is accommodated in a central four-helix bundle located in a hydrophobic pocket, with the six histidines bound to the copper pair This particular enzyme behaves as a catechol oxidase as it does not show monophenolase activity, and the o-diphenol binds to CuB [4,32] The most likely explanation for the lack of monophenolase activity of this PPO is related to the position of the bulky aromatic residue F261 In sweet potato o-diphenolase, F261 blocks access to CuA [4,26] This aromatic residue acts as a gate, controlling the accessibility of phenolic substrates to the hydrophobic pocket where the dinuclear copper center is found In addition, van der Waals interactions between this aromatic residue lining the hydrophobic cavity and the aromatic ring of phenolic substrates help to determine the affinity of substrates for the enzyme In wild-type and mutated mouse tyrosinase, it was proposed that the absence of this aromatic residue at the equivalent position may be the reason why it shows monophenolase activity, assuming that residue controls the access of monophenols to CuA [31] Although monophenols and o-diphenols could access CuB, F261 may block the re-orientation of monophenols toward CuA that is needed for its hydroxylation once is bound to CuB [32] It is very unlikely that minor details can be universally extrapolated to all tyrosinases and catechol oxidases from any source, but there is no doubt that this factor is important for accessibility to (or involvement of) CuA in the PPO active site in order for it to display monophenolase and o-diphenolase activity or just the latter activity For instance, all catechol oxidases from tomato, potato and beans have the aromatic residue at the equivalent position (Table 1) However, Streptomyces tyrosinases usually have the smallest residue, G, there In octopus hemocyanin, L2830 occupies the position of F261, and this may be responsible for the weak o-diphenolase activity detected in this protein, as an L residue blocks CuA less effectively than F Our results on the two PPOs found in R solanacearum are totally in agreement with this steric hindrance (Table 1) The product of the RSc1501 gene (NP_519622) has I294, a bulky but not aromatic residue, at the equivalent position followed by P295, a rigid residue It shows very low but measurable monophenolase activity The product of the RSc0337 gene (NP_518458) has in that place a small residue, A241, in agreement with the high tyrosine hydroxylase activity shown by this enzyme (Fig 9) This steric hindrance is one of the bases of the difference between monophenolases and o-diphenolases, but FEBS Journal 273 (2006) 257–270 ª 2005 FEBS ´ D Hernandez-Romero et al His 81 A novel tyrosinase from Rastonia solanacearum His 252 Ala 241 His 253 Cys 91 CuA His 93 CuB His 231 His 102 His 227 9A: NP_518458 (gene RSc 0337) His 153 Ile 294 Leu 306 His 307 CuA His 163 CuB His 283 His 172 His 279 9B: NP_519622 (gene RSc1501) Fig A plausible scheme for the conformation of the active site for the two PPOs presented here The basic structure with the six copper-binding H residues is drawn as described by Gerdemann et al.[4] for catechol oxidase Two main differences in R solanacearum PPOs are indicated First, the residue preceding H3B, which is H in NP_518458 and L in NP_519622 Second, the residue equivalent to the F261 of sweet potato catechol oxidase, responsible for accessibility to CuA, which is probably occupied by A in NP_518458 and I in NP_519622 it is not the only factor Other residues must be involved in the mechanism of catalysis It is also known that both PPO types, tyrosinases and catechol oxidases, show very different behavior with carboxylated and decarboxylated substrates According to Fig and the summary in Table 3, tyrosinase shows more affinity for and catalytic efficiency with carboxylated substrates, l-tyrosine vs tyramine and l-dopa vs dopamine The situation is the opposite for catechol oxidase The difference between carboxylated and decarboxylated substrates is related to the difference between monophenols and o-diphenols as favored substrate, and also related to the presence or absence of a seventh FEBS Journal 273 (2006) 257–270 ª 2005 FEBS histidine, adjacent to the sixth histidine involved in copper binding Preceding the sixth histidine (H3B according to the nomenclature used in [6]), at the end of the CuB-binding site, tyrosinase-like enzymes show an important variation Usually there is another H residue, but in some cases an L residue is found (for instance, in the NP_519622 protein from R solanacearum and in all animal tyrosinase-related proteins, Tyrp1 and Tyrp2 [6,31]) Accordingly, the amino-acid pair at that position is HH or LH The variant residue determines the putative interaction with the carboxylic group on the side chain of the phenolic substrate, and the affinity for the substrate All enzymes with the HH pair show high affinity for carboxylated substrates, but all enzymes with LH show higher affinity for decarboxylated substrates, as formerly reported [31] and confirmed in this work According to our proposal that the seventh histidine is not the proton acceptor residue necessary to withdraw the phenolic hydrogen for its co-ordination to the CuB [32], another residue must obey this role Its identity is not known Another proposed suggestion for that role is an E residue based on its proximity to the copper ions This is E236 in sweet potato catechol oxidase [26], equivalent to EB1-4 in the general terminology However, according to Table 1, this residue is conserved only in plant catechol oxidases Tyrosinases from Streptomyces have G at that position, animal tyrosinases generally have Q, and mushroom tyrosinase has I These data indicate that many efficient tyrosinases carry out the catalysis without this acidic residue, thus its requirement is very doubtful Rather than a proton acceptor contributing to catalysis, this E seems to be an obstacle to TH activity On the other hand, having monophenolase activity does not directly mean that this is the favored activity The formerly discussed three positions confirm that NP_518458 can act on carboxylated monophenols, but they are not enough to account for its high TH ⁄ DO ratio This special feature must be due to a unique residue(s) of this tyrosinase With regard to this, the pair of MM residues found just before the double HH at the CuB-binding site is particularly interesting It has recently been reported that, in copper protein type-1, axial methionines in positions close to the copper ion greatly affect the redox potential and catalytic efficiency [33] Extrapolation to copper type-3 is an appealing possibility Site-directed mutagenesis needs to be performed to clarify which factors are actually responsible for the catalytic properties of this PPO, and experiments on this are being carried out in our laboratory Apart from the interest of this novel tyrosinase as a model for the mechanism of catalytic cycles, and its 267 ´ D Hernandez-Romero et al A novel tyrosinase from Rastonia solanacearum physiological role in the pathogenic process, it may also be a novel biocatalyst useful in biotechnological applications that need a high monophenolase activity accompanied by low o-diphenolase activity In addition, this enzyme is quite resistant to temperature and chaotropic agents in comparison with tyrosinases from other sources Streptomyces glaucescens has a very labile enzyme, showing a half-life at 60 °C of  [8], but tyrosinase from R solanacearum is quite resistant to that temperature and SDS, although there are more resistant tyrosinases, such as the enzyme from Thermomicrobium roseum, which is almost unaffected by this temperature [34] Further studies are necessary to explore the possible biotechnological applications of these enzymes Experimental procedures Cell culture R solanacearum was grown in basal saline medium containing 15 mm (NH4)2SO4, 0.8 mm MgCl2, lm FeSO4, 0.2 mm CaCl2, lm Na2MoO4, lm MnCl2, 0.5% glycerol and 0.01% yeast extract in 50 mm sodium phosphate buffer, pH E coli was routinely grown in Luria–Bertani medium When required, media were supplemented with 50 lgỈmL)1 kanamycin, ampicillin or rifampicin, as necessary depending on the plasmid used and selection needed To obtain routine bacterial extracts, cells were grown in basal saline medium for 48 h to an absorbance of  1.2 and centrifuged at 5000 g for 10 The pellet was washed with 0.9% NaCl solution, resuspended in mL 0.1 m sodium phosphate, pH 7.0, containing 0.1 mm phenylmethanesulfonyl fluoride plus a : 500 dilution of ‘Protease inhibitor cocktail’Ò and disrupted by discontinuous sonication by using a Braun Labsonic sonicator for  The homogenate was centrifuged at 12 000 g for min, and the supernatant was used for purification and ⁄ or enzymatic activity determinations Reagents for cell culture media and enzymatic determinations were obtained from Sigma Co (St Louis, MO, USA) Protein determination Protein concentration was determined with the bicinchoninic acid assay Alternatively, A280 was used to follow the protein elution profile in chromatography purification columns Enzymatic determinations TH and DO activities were determined by monitoring, respectively, the oxidation of mm l-tyrosine or l-dopa to l-dopachrome at 475 nm (Fig 1B; e ẳ 3700 m)1ặcm)1), in 268 0.1 m sodium phosphate buffer The pH was 5.0 or 7.0 according to the activity and PPO enzyme assayed due to the different optimal conditions displayed by the activities of this micro-organism [17] Moreover, 0.05% SDS was added for the standard TH assay, and 0.02% SDS was added for DO activity For dopa titration, 50 lL 10 mm sodium periodate was added, and the increase in A475 immediately determined A small concentration of l-dopa was occasionally added as cofactor for TH activity when appropriate (detailed in results) Tyramine hydroxylase and dopamine oxidase were monitored in the same way, but dopaminochrome was determined (e ¼ 3100 m)1Ỉcm)1) In all cases, one unit was defined as the amount of enzyme that catalyses the appearance of lmol dopachrome ⁄ dopaminochrome per minute at 37 °C Enzyme purification The extracts were concentrated using 15-mL Amicon ultra Millipore centrifuge tubes of (cut-off 10 kDa) The preparations were loaded on a CM-Sephadex (Amersham Pharmacia Biotech, Amersham, Bucks., UK) column (3 cm diameter · 18 cm long) The column was eluted with 0.05 m sodium phosphate buffer, pH 7, until a volume approximately equal to the total volume of the column had been used Once nonbound proteins were eluted from the column, other proteins were eluted with a linear gradient of NaCl up to 1.5 m in the same buffer Fractions were assayed for PPO activity SDS/PAGE Enzyme purity was confirmed by SDS ⁄ PAGE [24] Analytical dissociating SDS ⁄ PAGE was performed using 9% acrylamide for the separating gel and 3% for the stacking gel The resolving buffer was Tris ⁄ HCl (pH 8.8), and the reservoir buffer was Tris ⁄ glycine (pH 8.3), both containing 0.1% SDS Samples were mixed in a : (v ⁄ v) ratio with sample buffer (0.18 m Tris ⁄ HCl, pH 6.8, 15% glycerol, 0.075% bromophenol blue, 7.5% 2-mercaptoethanol and 9% SDS) and heated at 95 °C for before application Electrophoresis was run at 20 °C and a constant current of 15 mA for 20 and 30 mA for  90 Protein bands were visualized by Coomassie Brilliant Blue staining Reagents for SDS ⁄ PAGE were obtained from Bio-Rad Laboratories (Richmond, CA, USA) Acknowledgements This work was supported in part by the project BIO2004-4803 from CICYT, Spain D.H.R was been supported by a financial grant associated with project BIO2001-0140 Special thanks go to Professor Boucher for supplying us with the sequenced strain FEBS Journal 273 (2006) 257–270 ª 2005 FEBS ´ D Hernandez-Romero et al References Mason HS (1956) Structures and functions of the phenolase complex Nature 177, 79–81 Van Gelder CW, Flurkey WH & Wichers HJ (1997) Sequence and structural features of plant and fungal tyrosinases Phytochemistry 45, 1309–1323 Solomon EI, Sundaran UM & Machonkin TE (1996) Multicopper oxidases and oxygenases Chem Rev 96, 2563–2605 Gerdemann C, Eicken C & Krebs B (2002) The crystal structure of catechol oxidase New insights into the functions of type-3 copper proteins Acc Chem Res 35, 183–191 Robb DA (1984) Tyrosinase In Copper Proteins and Copper Enzymes (Lontie R, ed.), Vol 2, pp 207–240 CRC Press, Boca Raton, FL ´ 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Hernandez-Romero et al the substrate analogue p-nitrophenol studied by NMR J Am Chem Soc 127, 567–575 33 Li H, Webb SP, Ivanivic J & Jensen JH (2004) Determinants of the relative reduction potentials of type-1 copper sites in proteins J Am Chem Soc 126, 8010–8019 34 Kong KH, Hong MP, Choi SS, Kim YT & Cho SH (2000) Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum Biotechnol Appl Biochem 31, 113–118 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS ... l-dopa as cofactor to eliminate the characteristic lag period of tyrosinases [8,20,21] Figure shows the rate of TH activity as a function of the concentration of l-dopa cofactor added to the assay.. .A novel tyrosinase from Rastonia solanacearum ´ D Hernandez-Romero et al A B C particular catechol to o-dopaquinone is also called dopa oxidase (DO) activity On the other hand, laccases... tyrosinase from Rastonia solanacearum environmental pH, may also affect the expression of the most appropriate enzyme Apart from the physiological roles and environmental advantages of having several