Báo cáo khoa học: Role of the C-terminal extension in a bacterial tyrosinase Michael Fairhead and Linda Thony-Meyer doc

Role of the C-terminal extension in a bacterial tyrosinase Michael Fairhead and Linda Tho ¨ ny-Meyer EMPA, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, St Gallen, Switzerland Introduction Tyrosinases and the related catechol oxidases (collec- tively termed polyphenol oxidases) comprise a family of binuclear copper enzymes found in many species of animals, plants, fungi and bacteria that use phe- nol-like starting materials to produce a variety of biologically important compounds, such as melanin and other polyphenolic compounds [1–3]. These type III copper proteins are capable of two activities: monophenolase or cresolase activity (EC 1.14.18.1) and diphenolase or catecholase activity (EC 1.10.3.1). Both activities result in the formation of reactive quinones, and these species are important intermedi- ates in the biosynthesis of compounds such as melanin. Given the ability of tyrosinases to react with phenols and its di-copper redox centres, they have been proposed for use in a variety of biotechnological, biosensor and biocatalysis applications [2]. One exam- ple includes tyrosinase immobilization as an electro- chemical biosensor for a range of phenolic compounds [4]. The enzyme can also react with tyrosine found on polypeptides, and the reactive quinones formed allow for protein cross-linking to chitosan films as well as protein-protein cross-linking [5,6]. The only available crystal structure of the tyrosin- ases comes from the secreted enzyme of Streptomyces castaneoglobisporus [7] tyrosinase. The structure shows the enzyme in complex with its accessory caddie protein (see below). The tyrosinase is predominately a-helical in structure and contains six histidine residues co-ordinating the two copper atoms that form the active site of the enzyme. With respect to its overall fold and active site architecture, the bacterial enzyme is strongly similar to the related enzyme catechol Keywords C-terminal domain; melanin; tyrosinase; Verrucomicrobium spinosum; zymogen Correspondence L. Tho ¨ ny-Meyer, EMPA, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, Lerchenfeldstrasse 5, St Gallen, CH-9014, Switzerland Fax: +41 44 071 274 7788 Tel: +41 44 071 274 7792 E-mail: linda.thoeny@empa.ch (Received 22 October 2009, revised 13 January 2010, accepted 22 February 2010) doi:10.1111/j.1742-4658.2010.07621.x The well studied bacterial tyrosinases from the Streptomyces sp. bacteria are distinguishable from their eukaryotic counterparts by the absence of a C-terminal extension. In the present study, we report that the tyrosinase from the bacterium Verrucomicrobium spinosum also has such a C-terminal extension, thus making it distinct from the Streptomyces enzymes. The entire tyrosinase gene from V. spinosum codes for a 57 kDa protein (full- length unprocessed form), which has a twin arginine translocase type signal peptide, the two copper-binding motifs typical of the tyrosinase protein family and the aforementioned C-terminal extension. We expressed various mutants of the recombinant enzyme in Escherichia coli and found that removal of the C-terminal extension by genetic engineering or limited tryp- sin digest of the pro-form results in a more active enzyme (i.e. 30–100-fold increase in monophenolase and diphenolase activities). Further studies also revealed the importance of a phenylalanine residue in this C-terminal domain. These results demonstrate that the V. spinosum tyrosinase is a new example of this interesting family of enzymes. In addition, we show that this enzyme can be readily overproduced and purified and that it will prove useful in furthering the understanding of these enzymes, as well as their biotechnological application. Abbreviations L-DOPA, L-3,4-dihydroxyphenylalanine; TAT, twin arginine translocase. FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2083 oxidase from sweet potato [8]; however, the plant enzyme is only capable of the diphenolase reaction (EC 1.10.3.1). The major distinguishing feature of the Strepto- myces sp. enzyme is the requirement for an accessory protein that is necessary for copper incorporation [1]. Several mutagenesis studies, as well as the crystal structure, have demonstrated the importance of this accessory ‘caddie protein’ for copper incorporation into the Streptomyces tyrosinase [7,9] and the expres- sion of active Streptomyces tyrosinase in either Escherichia coli or its native host requires the co-expression of the gene encoding this caddie pro- tein. This arrangement is entirely different from that of the eukaryotic enzymes, which are not known to require such a caddie protein and also have a C-ter- minal extension, the removal of which usually leads to a marked increase in activity [10]. Indeed, it is esti- mated that approximately 98% of the the tyrosinase present in mushrooms occurs in such a latent form [11]. However, the Streptomyces sp. tyrosinases may not be wholly representative of the bacterial form of these enzymes because the Rhizobium etli tyrosinase has been reported not to require a copper chaperone for activity [12]. Given their interesting properties and the wide poten- tial of these enzymes, there are few successful examples of recombinant production systems that provide high yields of pure enzyme, with most studies using the native Streptomyces sp. [13,14], Neurospora crassa [15] and Agaricus bisporus [2] enzymes. To cover this shortfall, we have cloned several uncharacterized tyrosinase genes from different bacterial species with the aim of identifying enzymes that have suitable characteristics for structure ⁄ function studies, as well as biotechnologi- cal applications. In the present study, we report the results obtained with the tyrosinase gene from Verrucomicrobium spinosum. Verrucomicrobium spinosum is part of the ubiquitous Verrucomicrobia phylum. These bacteria are found in a wide range of aquatic and terrestrial habitats [16,17]. Verrucomicrobium spinosum in particular is found in fresh water eutrophic (nutrient rich, oxygen poor) habitats and is capable of both aerobic and fer- mentative metabolism. This Gram-negative, yellow- pigmented bacterium is somewhat unusual as a result of the presence of numerous wart-like prosthecae appendages on its surface [17,18] and its compartmen- talized cytoplasm [19]. This bacterium is not known to normally produce melanin, and thus the presence of a tyrosinase gene in its genome was somewhat surprising because such genes are usually associated with black pigment formation in various bacterial and fungal species [20]. Results and Discussion Analysis of the V. spinosum tyrosinase gene region The V. spinosum tyrosinase gene is preceded upstream by a gene encoding a predicted laccase and followed downstream by a gene encoding a predicted b-sheet- rich protein for which we could find no obvious func- tion or homologue (Fig. 1A). This differs from the Streptomyces tyrosinase gene arrangement, where the tyrosinase is typically preceded by a gene encoding an accessory protein required for copper incorporation [1]. Given the absence of such a caddie protein gene upstream or downstream of the V. spinosum tyrosinase gene, we drew the conclusion that the V. spinosum tyrosinase does not require such a protein for copper insertion. The V. spinosum tyrosinase may therefore be similar to the aforementioned R. etli tyrosinase, which also has been reported not to require a copper chaper- one [12]. The presence of another multicopper oxidase- like laccase gene upstream of the tyrosinase gene is also interesting because laccases are known to be capable of synthesizing melanin, albeit usually from diphenols such as epinephrine and l-3,4-dihydroxy- phenylalanine (l-DOPA) [21]. Also present in the surrounding DNA sequence are several regions with homologies to the binding sites of E. coli RpoS and RpoD sigma factors, which are known to be involved in transcriptional regulation [22]. The predicted b-sheet-rich protein gene is fol- lowed by a region with a high probability of leading to an RNA secondary structure in the transcript, indica- tive of a site of transcription termination. The presence of these features may indicate that the tyrosinase gene is part of an operon. As stated in the Introduction, V. spinosum is not known to produce melanin under normal growth conditions. The laccase and ⁄ or tyrosinase are there- fore probably only synthesized under a specific set of circumstances or serve some alternative function to melanin production. We attempted to induce melanin synthesis by cultivating the V. spinosum bacterium on solid or in liquid media supplemented with excess copper or amino acids in an attempt to mimic con- ditions known to induce Streptomyces species tyro- sinases [23]. However, these experiments did not yield any detectable tyrosinase activities, as indicated by the lack of formation of any black pigments or Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho ¨ ny-Meyer 2084 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS monophenolase ⁄ diphenolase activities in bacterial extracts (data not shown). Features of the amino acid sequence of the V. spinosum tyrosinase The amino acid sequence of the full-length V. spino- sum pre-pro-tyrosinase (Fig. S1) can be divided approximately into three domains: a twin arginine translocase (TAT) signal peptide, a core domain con- taining the two copper-binding motifs and a C-termi- nal extension (Fig. 1B). The presence of a predicted TAT signal peptide at the N-terminus (amino acids 1–36) would suggest that the protein is exported to the periplasmic space of V. spinosum in an already folded form, as often found for metal-containing periplasmic proteins [24]. The presence of this signal peptide is in agreement with the fact that the Streptomyces tyrosin- ases are also secreted via the TAT secretion pathway [25]. Also present in the sequence are the two copper A (amino acids 86–96) and copper B (amino acids 258–294) binding motifs common to most tyrosinase sequences [3] that contain five of the six copper-bind- ing histidine ligands. The sixth histidine ligand found in tyrosinases typically occurs before the copper A motif. From sequence alignments, we suggest that this ligand is most likely histidine 80 in the V. spinosum tyrosinase. Another motif, which is present not only in tyrosinases, but also in the oxygen transporting haemocyanin proteins, is the PYWDW (amino acids 118–122) and has been hypothesized to be involved in oxygen binding [26]. Previous sequence analysis in other studies has dem- onstrated the presence of a conserved Yx(Y ⁄ F) motif in the C-terminal domains of both the Streptomyces type tyrosinases and processed eukaryotic tyrosinases and haemocyanins [10]. This motif can also be seen to be present in the V. spinosum tyrosinase (Figs 1B and S1). It has been hypothesized, with support from the crystal structure of catechol oxidase, that the tyrosine residue(s) in this motif form a hydrogen-bonding network to a conserved arginine residue close to the N-terminus that stabilizes the mature, processed form of polyphenol oxidases [8,10]. A homologue of this arginine residue is also present in V. spinosum tyrosinase (Arg40) (Figs 1 and S1). Another notable feature of the V. spinosum tyrosi- nase sequence is the presence of the proteins only cys- teine residue at position 84. A cysteine at this position is also found in some other eukaryotic tyrosinases and plant catechol oxidases. This cysteine may be of functional importance because it has been shown to form a novel alkane-thiol bond to one of the copper ligand histidine residues in the structure of the related sweet potato catechol oxidase [8]. The equivalent cysteine and bond are absent in the structure of S. cas- taneoglobisporus tyrosinase [7]. Indeed, Streptomyces A Copper binding motif TAT signal peptide 1–36 Pre-pro-tyrosinase 518 amino acids Core domain 37–357 C-terminal extension amino acids 358–518 Copper binding motif Arg40 Phe453 Cys84 Tyr349 Tyr347 Copper binding motif Pro-tyrosinase 481 amino acids Core domain 36–357 C-terminal extension amino acids 358–518 Copper binding motif Arg40 Phe453 Cys84 Tyr349 Tyr347 Copper binding motif Core tyrosinase 320 amino acids Core domain 36–357 Copper binding motif Arg40 Cys84 Tyr349 Tyr347 Copper binding motif Trypsinisedpro-tyrosinase 332 amino acids Core domain 36–370 Copper binding motif Arg40 Cys84 Tyr349 Tyr347 Lys370 Ala36 Ala36 Ala36 Val357 Phe518 B Laccase Tyrosinase β -sheet protein Fig. 1. Overview of the tyrosinase gene and surrounding genes in the genome of V. spinosum. (A) Showing the tyrosinase gene and those in its immediate vicinity in the V. spinosum genome. Triangles indicate regions with homology to the binding sites of the E. coli RpoS and RpoD regulatory proteins; the octagon shows the position of a region predicted to have a high probability of RNA secondary structure, which is indicative of a termination transcript. (B) An overview of the pre-pro-tyrosinase, pro-tyrosinase and core-tyrosinase constructs and their notable features. M. Fairhead and L. Tho ¨ ny-Meyer Recombinant V. spinosum tyrosinase FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2085 sp. tyrosinases contain no cysteine residues at all [27]. However, experimental evidence does demonstrate the presence of such a bond in N. crassa tyrosinase [15] and in molluscan haemocyanins [28]. The arrangement of a core tyrosinase domain followed by a C-terminal extension (Fig. 1B) is similar in design to mushroom tyrosinase and plant polyphenol oxidases [10]. The mushroom C-terminal domain can be removed by proteolysis or denatured by SDS, leading to an activation of the enzyme [11,29]. By contrast, the Streptomyces type tyrosinases have no such C-terminal extension after the core tyrosinase domain [1]. One proposed function of the C-terminal extension in plant and fungal polyphenol oxidases is a role in membrane binding, making them similar to the mam- malian tyrosinases, which have a single transmembrane domain [27]. However, it is considered that the plant forms are not integral membrane proteins because they can be released in an active form from the membrane by sonication, proteolysis or treatment with mild deter- gents [30,31]. Thus, whether the C-terminal domain in the plant and fungal enzymes has a purely inhibitory function and ⁄ or a role in membrane binding is unclear at present. With regard to V. spinosum pro-tyrosinase, sequence analysis of the C-terminal domain, and indeed of the entire sequence, suggested that no trans- membrane helices were present, as also demonstrated by the fact the enzyme is produced in a soluble form in E. coli. Recombinant expression of V. spinosum tyrosinase in E. coli To study the properties of the V. spinosum tyrosinase, we created a range of constructs (Table 1) for recombi- nant expression of the pre-pro-tyrosinase, the pro- tyrosinase and the core tyrosinase (Fig. 1B). It can be seen from Fig. 2 that E. coli cells transformed with plasmids containing either the pre-pro-tyrosinase or the pro-tyrosinase tyrosinase constructs (Fig. 2B, C) produced a black pigment when streaked onto M9 agar plates containing tyrosine and copper, whereas a strain lacking a tyrosinase construct remained white (Fig. 2A). The activity observed on the M9 agar plates was found to correlate with over-expression of the various proteins in liquid media. It can be seen from the gel presented in Fig. 3A that bands are present in samples of lysate of E. coli cells transformed with plasmids encoding the different tyrosinase variants. These bands correspond to the calculated molecular masses of the respective polypeptides (Table 1), namely 57 kDa for pre-pro-tyrosinase (lane 4) and 53.4 kDa for pro-tyros- inase (lane 3). The different constructs were expressed at different levels, with an increase in expression occur- ring when the putative N-terminal TAT signal peptide was removed (Fig. 3A, lanes 3 and 4). We found it necessary to express all the tyrosinase constructs in an apo-form, by growing and inducing Table 1. List of active constructs produced in the work and their features. ND, not determined; NA, not applicable. Name (plasmid) Mutations or modifications Calculated molecular mass (kDa) a Determined molecular mass b pI a Extinction coefficient 280 nm (m M )1 Æcm )1 ) a Purpose Pre-pro-tyrosinase (pMFvppt) Amino acids 1–518 57.005 ND 7.2 91.9 Full-length tyrosinase gene from V. spinosum Pro-tyrosinase (pMFvpt) Amino acids 36–518 with non-original methionone start codon 53.500 53.501 6.9 86.4 Removal of TAT signal pepetide from pro-tyrosinase gene for cytosolic expression Trypsinized pro-tyrosinase (NA) Amino acids 36–370 37.873 37.874 8.1 80.9 Removal of c-terminal extension via trypsin for improved activtiy Core tyrosinase (pMFvct) Amino acids 36–357 with non-original methionone start codon 36.507 36.506 7.1 80.9 Removal of c-terminal extension for improved activity Pro-tyrosinase F453A (pMFvptf2a) Pro-tyrosinase with phenylalanine 453 mutated to alanine 53.4 ND 6.9 86.4 To check whether this residue performs a ‘gatekeeper’ function at the tyrosinase active site a Values calculated using PROTPARAM (24). b Molecular mass determined by MS. Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho ¨ ny-Meyer 2086 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS the transformed cells in media prepared using Milli-Q water (Millipore, Billerica, MA, USA) and lacking added copper. This was necessary because, otherwise, a black pigment was produced during incubation. This pigment was found to inhibit the growth of E. coli and to foul protein purification columns, both of which resulted in a low protein yield. This problem was par- ticularly acute with the highly active core tyrosinase. The formation of a black pigment (presumably mela- nin) was most likely a result of the action of the expressed tyrosinase on the tyrosine present in the pep- tone or N-Z-amine Ò (Sigma-Aldrich, Buchs, Switzer- land) that was added to the expression medium as an external source of amino acids to aid recombinant protein production. Provided the precaution of not supplying copper to the medium was taken, we found that soluble protein could be obtained for all the described constructs. In experiments with the pre-pro-tyrosinase construct, we did not obtain sufficient amounts of protein for purification. We also attempted to isolate the protein from the E. coli periplasm but could not find any evi- dence of activity, indicating a lack of export of the protein. It could be that the E. coli TAT system is unable to recognize the V. spinosum export signal peptide. When designing tyrosinase constructs without the predicted N-terminal signal peptide (amino acids 1–36), we retained amino acid 36, an alanine, rather than using amino acid 37, a lysine, because it is known that, after a post-translational processing of the N-terminal methionine, which often occurs for proteins expressed in E. coli, according to the N-end rule, a newly-created N-terminal lysine would result in a very short protein half-life, whereas an N-terminal alanine would be fine [32]. The recombinant pro-tyrosinase was expressed and purified with final yields of approximately 20 mgÆL )1 of pure protein. Subsequent analytical gel filtration of the purified pro-tyrosinase showed a single peak corre- sponding to a monomer (Fig. S2). The mass of the purified protein determined via MS (53 501 kDa) corresponded closely to the expected full-length pro- tyrosinase (53 500 kDa) assuming the removal of the N-terminal methionine. Reconstitution of recombinat V. spinosum tyrosinase with copper The holo-forms of tyrosinase were obtained after puri- fication by adding copper to a three-fold molar excess, and samples were subsequently exhaustively dialysed in an attempt to remove any nonspecifically bound cop- per. The final copper content of the dialysed samples was then determined (Table 2). Although pro-tyrosi- nase was found to be nearly fully loaded with copper using this method (1.8 molar equivalents), the core tyrosinase and pro-tyrosinase F453A mutant were found to be significantly under-loaded (1.4 and 1.2 molar equivalents respectively). It is possible that the protocol used was not optimal for copper incorporation into these variants (see Experimental A CD E B Fig. 2. Melanin formation on tyrosine con- taining solid media by E. coli cells express- ing V. spinosum tyrosinase constructs. (A) Escherichia coli transformed with vector containing no insert (pQE-60); (B) E. coli transformed with pMFvppt (pre-pro-tyrosi- nase); (C) E. coli transformed with pMFvpt (pro-tyrosinase); (D) E. coli transformed with pMFvct (core tyrosinase); (E) E. coli trans- formed with pMFvptf2a (pro-tyrosinase F453A). M. Fairhead and L. Tho ¨ ny-Meyer Recombinant V. spinosum tyrosinase FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2087 procedures) and, indeed, it has been reported that incubation at pH 6 may result in higher levels of cop- per reconstitution than at pH 8 [33,34]. We are cur- rently investigating this possibility. In addition, despite extensive dialysis of reconsti- tuted samples, it cannot be excluded that some of the copper is nonspecifically bound to the protein. We have found, however, that attempts to remove any such copper ions with low concentrations of the chelat- ing agent EDTA (100 lm) resulted in a complete loss of activity and detectable copper. As an alternative to copper reconstitution of the purified proteins, we also attempted to grow bacteria in minimal media contain- ing copper as a means of producing holo protein directly. However, we found that the omission of an external amino acid source such as N-Z-amine led to very low levels of tyrosinase expression, as well as low cell densities, meaning that the purification of holo protein in this way was impracticable. C-terminal processing by trypsin As noted above, the C-terminal extension found in the latent form of mushroom tyrosinase has been shown to be inhibitory to activity, and its removal by serine proteases such as subtisilin results in an activation of the enzyme, similar to the protease zymogen system found for many digestive enzymes, such as trypsin [11]. The related plant catechol oxidase enzymes also have similar C-terminal extensions [10]. Sequence analysis suggested that this may also be the case for the V. spinosum enzyme (see above). We therefore used trypsin digestion to determine whether a smaller, more active fragment could be produced from purified pro- tyrosinase. The gel in Fig. 3C shows that trypsin diges- tion indeed yielded a smaller stable fragment, which was subsequently found to be far more catalytically active than the original pro-tyrosinase (Table 3). The stability of the smaller trypsinized fragment, even after 24 h of incubation with trypsin, suggests that this is a highly ordered domain with no accessible cleavage sites for trypsin. This interpretation corresponds to the pro- posal that the C-terminal extension of eukaryotic poly- phenol oxidases (i.e. tyrosinase and plant catechol oxidases) is highly disordered [10] compared to the corresponding core oxidase domains containing the two copper-binding motifs. These disordered domains would thus be more susceptible to proteolysis than the more ordered stable core domains of the enzymes. High levels of disorder in the pro-domain are also present in zymogens such as in procathepsin K [35] and probably represent an important feature in the activation mechanism of these enzymes. The fact that A B C Fig. 3. (A) SDS-PAGE of cells expressing the tyrosinase constructs. Lane 1, lysate from cells transformed with pMFvptf2a (pro-tyrosi- nase F453A); lane 2, lysate from cells transformed with pMFvct (core tyrosinase); lane 3, lysate from cells transformed with pMFvpt (pro-tyrosinase); lane 4, lysate from cells transformed with pMFvppt (pre-pro-tyrosinase); lane 5, lysate from control cells transformed with pQE-60 containing no insert. (B) SDS-PAGE of purified and trypsinized tyrosinases. Lane 1, purified pro-tyrosinase; lane 2, puri- fied core tyrosinase; lane 3, purifed pro-tyrosinase F453A mutant; lane 4, trypsinized pro-tyrosinase; lane 5, trypsinized core tyrosi- nase. (C) SDS-PAGE showing time course of proteolysis of pro-tyrosinase by trypsin. Lane 1, pro-tyrosinase after 24 h of incu- bation at room temperature; lane 2, trypsin after 24 h of incubation at room temperature; lane 3, pro-tyrosinase plus trypsin after 0 h at room temperature; lane 4, pro-tyrosinase plus trypsin after 1 h at room temperature; lane 5, pro-tyrosinase plus trypsin after 4 h at room temperature; lane 6, pro-tyrosinase plus trypsin after 24 h at room temperature. M, Molecular mass markers. Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho ¨ ny-Meyer 2088 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS the pro-tyrosinase exhibits some low levels of catalytic activity also suggests some mobility between the core tyrosinase domain and the C-terminal extension (Table 3). Recombinant core tyrosinase To further asses the functional importance of the C-terminal extension, we created a shortened form of the V. spinosum tyrosinase, using the presence of the conserved YX(Y ⁄ F) motif as a guide. The resulting construct was readily overexpressed in the E. coli cytoplasm (Fig. 3A, lane 4) and found to be highly active after loading with copper compared to the pro-tyrosinase form (Table 3). We also treated the mature (i.e. copper-containing) tyrosinase with trypsin and found that the trypsinized recombinant core tyrosinase (Fig. 3B, lane 5) exhibited no apparent size difference compared to the un-trypsi- nized preparation (Fig. 3B, lane 2) but appeared to be smaller than the trypsinized pro-tyrosinase (Fig. 3B, lane 4). Determination of the mass of the proteins by MS revealed masses of 36 506 kDa (recombinant core tyrosinase) and 37 874 kDa (trypsinized pro-tyrosi- nase) corresponding to a C-terminal amino acid of Val357 and Lys370, respectively. Gel filtration revealed that both proteins also exist in solution, similar to pro- tyrosinase, as monomers (Fig. S2). The results obtained in the present study suggest that the C-terminal extension has no role in copper insertion like the Streptomyces sp. ‘caddie’ protein because the recombinant core tyrosinase enzyme was found to be readily reconstituted with copper, as indi- cated by its high activity and subsequent analysis of its copper content (Table 2). This correlates with the results obtained using apo-forms of mature tyrosinase from both N. crassa [36] and A. bisporus [37], which could also be readily reconstituted with copper. This is in contrast to the results obtained with the Streptomy- ces sp. enzyme [38,39], which has an absolute require- ment for the accessory caddie protein for copper incorporation. Furthermore, the results obtained in the present study are in agreement with the previously noted finding that, in the gene region around the V. spinosum tyrosinase, no gene encoding a caddie-like protein is present (Fig. 1A). Because the pro-tyrosinase form contains no predicted transmembrane helices and is indeed fully soluble in E. coli (see above), we suggest that the C-terminal extension in this case has a purely inhibitory function and neither a significant role in stabilizing the enzyme, nor a chaperone-like function during folding, as has been proposed for other N-terminal ⁄ C-terminal zymogen-like systems [40]. It remains to be determined whether this is also the case for other pro-tyrosinase forms. Stability of the tyrosinase forms to chemical denaturation To characterize the domain structure of the V. spino- sum tyrosinase in more detail, we determined protein stability by recording protein unfolding via fluores- cence spectroscopy when increasing amounts of guani- dine hydrochloride (GdnCl) were present. The determined unfolding curves (Fig. S3) appeared to show two apparent transitions for holo pro-tyrosinase and one for either holo trypsinized pro-tyrosinase or the holo recombinant core tyrosinase. However, the unfolded proteins were not found to refold once Table 2. Stability and determined copper content of the tyrosinase enzymes. ND, not determined. Enzyme GdnCl concentration ( M) at 50% unfolded a Molar equivalents of copper Holo pro-tyrosinase 2.2 1.8 Apo pro-tyrosinase 1.3 0.01 Holo trypsinized pro-tyrosinase 3.3 b 1.8 b ⁄ 1.5 c Apo tyrpsinized pro-tyrosinase 2.0 0.4 Holo core tyrosinase 2.9 1.4 Apo core tyrosinase 1.8 0.02 Holo pro-tyrosinase F453A ND 1.2 Apo pro-tyrosinase F453A ND 0.1 a Protein solutions (0.1 mgÆmL )1 ) were incubated for 24 h at room temperature in 10 m M Tris-HCl (pH 8) containing 0–6 M GdnCl before measurements were made (for details, see Experimental procedures). b Copper content and stability determined with trypsi- nized holo pro-tyrosinase. c Copper content determined by reconsti- tuting trypsinized apo pro-tyrosinase. Table 3. Monophenolase and diphenolase activities of the tyrosi- nase enzymes. Activity of the various constructs ⁄ mutants towards the model substrates L-tyrosine and L-DOPA (n = 3 for all determi- nations). Enzyme L-tyrosine L-DOPA V max a K m (lM) V max a K m (mM) Pro-tyrosinase 5.8 ± 0.6 421 ± 43 4.7 ± 0.3 7.0 ± 0.7 Trypsinized pro-tyrosinase b 325 ± 8 258 ± 6 565 ± 20 7.9 ± 0.5 Core tyrosinase 148 ± 4 280 ± 15 230 ± 7 7.6 ± 0.3 Pro-tyrosinase F453A 16 ± 0.9 808 ± 66 14 ± 0.2 6.4 ± 0.4 a Units = lmol dopachromeÆmin )1 ÆmgÆprotein )1 . b Values deter- mined for trypsinized holo pro-tyrosinase. M. Fairhead and L. Tho ¨ ny-Meyer Recombinant V. spinosum tyrosinase FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2089 denatured and, thus, the apparent shapes of the unfolding curves should not be over interpreted. The use of the concentration of GdnCl at 50% unfolded as a simple measure of the change in stability between the various tyrosinase forms allows some conclusions to be drawn (Table 2). The values show that the incorpora- tion of copper into either pro-tyrosinase, trypsinized pro-tyrosinase or recombinant core tyrosinase signifi- cantly increases the overall stability of the protein. It was also apparent that the C-terminal extension of pro-tyrosinase reduces its overall stability in either the holo- or apo-forms of the enzyme. The negative effect on stability as a result of C-terminal extension would suggest this domain is less stable than the core domain of the enzyme, which correlates with the results obtained with trypsin digestion. It can also be seen from Table 2 that the recombinant core domain tyrosi- nase appears to be less stable than the trypsinized pro- tyrosinase; this could be a result of its reduced copper content. Alternatively, it may be that the recombinant core tyrosinase C-terminal extension is slightly too short for optimal stability and that residues after the YX(Y ⁄ F) motif also play a role in protein stability. Mono- and diphenolase activities of the recombinant tyrosinases When we measured activities towards either l-tyrosine or l-DOPA of pro-tyrosinase, a major increase in activity upon removal of the C-terminal extension by trypsin was found, namely an approximately 50-fold increase in mono- and a 100-fold increase in dipheno- lase activitiy (Table 3). There was also a less significant lowering in the K m value for l-tyrosine upon removal of the C-terminal extension (i.e. from 421 to 258 lm). The activities of the trypsinized pro-tyrosinase towards l-tyrosine or l-DOPA was found to be approximately twice that of the recombinant core tyrosinase, although the K m for both substrates is almost identical. The increased level of activity is prob- ably a result of the higher copper content of the trypsi- nized pro-tyrosinase (Table 2). The actual activities of the trypsinized pro-tyrosinase and recombinant core tyrosinase towards l-DOPA (i.e. 565 and 230 lmol dopachromeÆmin )1 Æmg protein )1 , respectively) compare favourably with the activities reported for Strepto- myces antibioticus tyrosinase, which are 1000 dopa- chromeÆmin )1 Æmg protein )1 [41]. The K m values for these two preparations towards l-DOPA (7.9 and 7.6 mm, respectively) are also similar to those report- ed for the S. castaneoglobisporus enzyme (8.1 mm) but substantially higher than that reported for the A. bisporus enzyme (0.8 mm) [42]. However, the K m values for l-tyrosine (258 and 280 lm, respectively) were similar to that of the A. bisporus enzyme (270 lm) [42]. Role of Phe453 in the pro-tyrosinase C-terminal The inhibitory effect of the C-terminal extension found in some plant polyphenol oxidases has been hypothe- sized to be a result of the presence of an amino acid that occludes the active site. This idea has been proposed because of similarities in the structures of the C-terminals of the related family of haemocyanins to plant polyphenol oxidases [3]. The crystal structure of octopus haemocyanin shows that a leucine (Leu2830) residue is present near the active site and acts as a ‘blocking residue’ [43]. This ‘blocking residue’ prevents substrate molecules from entering the active site, although oxygen can freely diffuse in and out, allowing oxygen transport to be the primary function of this protein. However, upon denaturation with SDS or proteolysis, it has been observed that tyrosinase-like activities can be introduced into haemocyanins and this has been proposed to occur via movement of the ‘blocking residue’ [44]. A leucine or similar hydropho- bic residue in an equivalent position has also been demonstrated to be present by sequence alignments of plant polyphenol oxidases [3]. In the case of the catechol oxidase from Ipomea, molecular modelling of the C-terminal domains was used to propose Leu439 as the ‘blocking residue’ [45]. Using a similar process of sequence alignment, we hypothesized that the functional equivalent of this blocking residue in V. spinosum pro-tyrosinase is Phe453. Thus, we constructed a pro-tyrosinase mutant carrying an alanine at this position, F453A. Curi- ously, an increase in protein expression was obtained for this mutant tyrosinase similar to that obtained when the entire C-terminal extension was removed (i.e. that of the core tyrosinase; Fig. 3B, lanes 1–3). It can be seen from the results shown in Table 3 that this variant had a higher activity than wild-type pro- tyrosinase, as would be expected if the amino acid residue at this position has the aforementioned block- ing function. However, the level of increase is very modest (approximately three-fold) compared to a vari- ant in which the C-terminal domain was removed completely by trypsin digest (50- to 100-fold). How- ever, it should be noted that copper analysis revealed that this mutant was very underloaded with copper (only 1.2 equivalents per mole rather than the expected 2). It could be reasonably expected that a higher level of loading would allow much greater levels of activity. Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho ¨ ny-Meyer 2090 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS The importance of Phe453 in pro-tyrosinase is also indicated by the fact that we could not induce wild- type pro-tyrosinase to form its active oxy complex, as indicated by its absorbance spectrum, whereas the F453A mutant, similar to the recombinant core tyrosi- nase, readily formed this complex (Fig. S4). These results suggest that the Phe453 residue is in the close vicinity of the enzyme active site and plays some role in oxygen binding. Nonfunctional tyrosinase mutants To further investigate the function of various other amino acids in V. spinosum tyrosinase, we also con- structed two further mutants. The importance of Arg40 as a potential residue interacting with Tyr347 and Tyr348 was tested by changing the arginine to an alanine. However, the mutation abolished the expres- sion of recombinant protein completely (not shown), which could indicate this residue is vital for protein stability. We also attemted to test whether Cys84 of the V. spinosum tyrosinase has a similar role in forming an alkane thiol bond, as has been shown for the sweet potato catechol oxidase [8] or the N. crassa tyrosinase enzyme [15]; therefore, this residue was mutated to a serine in the pro-tyrosinase. Unlike in A. oryzae, where a similar mutation resulted in a loss of activity but not of expression [46], we found that this mutation resulted in a complete loss of detectable protein. This suggests that the residue is essential for correct folding and expression of the enzyme. This appeared to contradict the results obtained with the A. oryzae enzyme; how- ever, it should be noted that this is a unique tyrosinase that has a novel acid-induced self-activation mecha- nism [47]. Furthermore, it has been shown to change from a tetramer in the pro-form to a disulfide-linked dimer in the mature form. Because the V. spinosum pro-tyrosinase, its trypsinized form and the recombinat core domain were all found to be monomeric, they are probably not directly comparable to the A. oryzae enzyme (Fig. S2). Verrucomicrobium spinosum tyrosinase as an alternative model bacterial enzyme In summary, we present a system that allows the expression of high levels of a novel bacterial tyrosi- nase. This system has the advantage of an accessory copper chaperone not needing to be expressed for copper reconstitution because the protein can be expressed in the apo-form and reconstituted after purification. The expression and purification of the apo-form prevents melanin formation during culture growth, which greatly simplifies downstream process- ing and improves protein yields. The resulting enzyme preparations have been demonstrated to have high lev- els of tyrosinase activities provided the inhibitory C-terminal domain is removed either by proteolysis or recombinant expression. The recombinant V. spinosum tyrosinase constructs should prove useful for the investigation of non-streptomyces type tyrosinases and may also allow the determination of a crystal structure of a tyrosinase in its low activity pro-form as well as the solution of a structure that is not in complex with a caddie protein. Experimental procedures Materials Chemicals and proteins were purchased from Sigma- Aldrich, molecular biology reagents from Fermentas GmbH (Le Mont-sur-Lausanne, Switzerland) and oligonucleotides from Microsynth AG (Balgach, Switzerland). Chromatogra- phy resins and columns were purchased from GE Health- care Europe GmbH (Bjo ¨ rkgatan, Sweden). Molecular biology and molecular cloning Verrucomicrobium spinosum (strain No. 4136) was obtained from DSMZ GmbH (Braunschweig, Germany) and cul- tured under the recommended conditions [48]. Primers Ver- rucFP01 and VerrucRP01 were used to amplify the tyrosinase gene (Pubmed Locus Tag VspiD_010100001190) and were designed using the draft genome from TIGR (Project ID: 10620). The full-length gene was then cloned into the BamHI and HindIII sites of pUC18 and the sequence verified using the Synergene Biotech GmbH (Zurich, Switzerland) sequencing service. Mutants were made using standard PCR techniques or QuikchangeÔ (Stratagene, La Jolla, CA, USA) mutagenesis using the primers listed in Table S1. Protein expression For protein expression, the full-length tyrosinase insert or mutants thereof were sub-cloned into the EcoRI and HindIII sites of the pQE60 vector (Qiagen AG, Hom- brechtikon, Switzerland) using the VerrucRBSFP01 ⁄ VerrucRP01 or VerrucRBSFP02 ⁄ VerrucRP01 primer pairs. The resulting plasmids (Table 1) were transformed into E. coli strain DH5a. Constructs were tested for melanizing activity by streaking transformed cells onto M9-agar plates [19] containing 100 lm CuSO 4 ,1mm isopropyl thio- b-d-galactoside, 100 lgÆmL )1 ampicillin, 1% glycerol and 0.5 mgÆmL )1 (2.76 mm) l-tyrosine. The plates were then M. Fairhead and L. Tho ¨ ny-Meyer Recombinant V. spinosum tyrosinase FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2091 incubated at 37 °C overnight and visually checked the next day for the formation of melanin. For 1 L scale expression of the pro-tyrosinase and its F453A mutant, a 30 mL overnight culture was grown from a single transformant in LB [49] + 1% glucose + ampicil- lin 100 lgÆmL )1 . The overnight culture was used to inocu- late (1 : 50) 2 · 500 mL of M9 + medium, containing: M9 salts, 1% peptone, 1% glycerol and 1% glucose, 100 lm calcium (CaCl 2 ), 2 mm magnesium (MgSO 4 ), 100 lm thia- mine and 100 lgÆmL )1 ampicillin in 2 · 2 L Erlenmeyer flasks. This culture was grown at 37 °C with shaking at 180 r.p.m. for 4–5 h, D 600  0.5, then 1 mm isopropyl thio- b-d-galactoside and 100 lgÆmL )1 ampicillin was added and growth continued for another 20 h. Expression of the recombinant core domain of tyrosinase was performed using modified autoinduction media [50]. A 30 mL overnight culture was grown from a single trans- formant in LB + 1% glucose + ampicillin 100 lgÆmL )1 . The overnight culture was used to inoculate (1 : 50) 2 · 500 mL of auto induction media: 1% N-Z-amine, 0.5% yeast extract, 25 mm Na 2 HPO 4 ,25mm KH 2 PO 4 ,50mm NH 4 Cl, 5 mm Na 2 SO 4 , 1% glycerol, 0.4% lactose, 0.5% glucose, 100 lm CaCl 2 ,2mm MgSO 4 , 100 lm thiamine and 100 lgÆmL )1 ampicillin in 2 · 2.5 L full baffle Tunair flasks (Shelton Scientific, Shelton, CT, USA). This culture was grown at 37 °C with shaking at 160 r.p.m. for 24 h. Protein purification Cells were harvested by centrifugation and washed in 0.1 m Tris-HCl (pH 8). The washed cell pellet was resuspended using 2 mL of 0.1 m Tris-HCl (pH 8) per gram wet weight of cells, to which lysozyme was added to 1 mgÆmL )1 . Cells were incubated for 1 h on ice and then frozen at )80 °C. Cells were then thawed and sonicated with a Branson soni- fier cell disruptor (Branson Ultrasonics Corp., Danbury, CT, USA), equipped with a 13 mm tip on 50% power using five 20 s bursts. The sample was then centrifuged at 50 000 g for 30 min. To the soluble fraction, 0.6 g ⁄ mL of NH 4 SO 4 was then added and the sample centrifuged at 50 000 g for 30 min. The resulting pellet was dissolved in 20 mL of 0.1 m Tris-HCl (pH 8) and dialysed against 5 L of 10 mm Tris-HCl (pH 8) for 2 h, at which point the buf- fer was exchanged and dialysis continued overnight. The dialysed sample was then centrifuged at 50 000 g for 30 min. The desalted sample was then passed over a 160 mL bed volume Q-Sepharose fast flow column (GE Healthcare Europe GmBH) and the unbound fraction con- taining tyrosinase collected, running 10 mm Tris-HCl buffer (pH 8). Tyrosinase containing fractions were then pooled and concentrated to 5 mL and loaded onto Superdex 75 16 ⁄ 60 gel filtration column (GE Healthcare Europe GmBH), 120 mL bed volume, running 10 mm Tris- HCl + 0.1 m NaCl buffer (pH 8). Tyrosinase containing fractions were then pooled concentrated to 10 mgÆmL )1 and stored at –80 °C in 100 lL aliquots. All purification steps were performed using an A ¨ KTA purifier 100 FPLC (GE Healthcare Europe GmbH). The calculated extinction coefficients at 280 nm were used to measure the concentra- tion of the purified proteins (Table 1). Size determination For analytical gel filtration, a Superdex 75 16 ⁄ 60 column was used, 120 mL bed volume, running 10 mm Tris- HCl + 0.1 m NaCl buffer (pH 8). A calibration curve for size determination was made using blue dextran (2 MDa) and the proteins: horse heart cytochrome c (12.4 kDa), horse heart myoglobin (17 kDa), bovine b-lactoglobulin (35 kDa), ovalbumin (44.3 kDa) and bovine serum albumin (67 kDa) (Fig. S2). The sizes of purified proteins was also determined using the mass MS service of the ETH func- tional genomics centre Zurich (http://www.fgcz.ethz.ch/). Enzyme assay Kinetic characterization of l-tyrosine and l-DOPA oxidation was measured by dopachrome formation [51] at 475 nm using a molar extinction coefficient of 3600 M )1 Æcm )1 at 25 °Cin 3 mL of 0.1 m potassium phosphate buffer (pH 6.8) using a stirred Peltier assembly, with the spectra being monitored on a Cary 50 bio UV ⁄ visible spectrophotometer (Varian Inc., Zug, Swizerland). Kinetic parameters were calculated using prism 5 (GraphPad Software Inc., San Diego, CA, USA). Bioinformatics The molecular mass and theoretical extinction coefficient of the various proteins were calculated using the protparam tool available through the ExPasy server (http://www.exp- asy.ch/tools/protparam.html) [52]. The signalP server was used for signal peptide prediction (http://www.cbs.dtu.dk/ services/SignalP/) [53]. Copper reconstitution Purified apo-tyrosinase was reconstituted with copper by mixing an aliquot of protein ( 10 mg) with an equal vol- ume of 10 mm Tris-HCl (pH 8), containing a three-fold molar excess of CuSO 4 in a final volume of 1 mL. The sam- ple was incubated on ice for 1 h and then dialysed twice against 1 L of 10 mm Tris-HCl buffer (pH 8). Copper analysis The copper concentration of the protein samples was mea- sured using a slight modification of the biquinoline method [54]. Briefly 100 lLof10mgÆmL )1 protein sample was added to 0.2 mL of 0.1 m sodium phosphate Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho ¨ ny-Meyer 2092 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS [...]...M Fairhead and L Thony-Meyer ¨ buffer + 10 mm ascorbate (pH 6) To this, 0.7 mL of glacial acetic acid containing 0.5 mgÆmL)1 of 2,2-biquinoline was added The mixture was incubated for 10 min at room temperature and A5 46 was measured, using water as a reference A standard curve using 0–165 lm CuCl2Æ2H2O was also made and gave a calculated e for the copper biquinoline complex of 5982 m)1Æcm)1 Trypsinization... bisporus) tyrosinase by serine proteases J Agric Food Chem 47, 3509–3517 Cabrera-Valladares N, Martinez A, Pinero S, LagunasMunoz VH, Tinoco R, de Anda R, Vazquez-Duhalt R, Bolivar F & Gosset G (2006) Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase Enzyme Microb Technol 38, 772–779 Kohashi PY, Kumagai T, Matoba Y, Yamamoto A, Maruyama... Inc.) at 25 °C, with excitation at 285 nm and emission at 300–400 nm, and the unfolding curve was calculated [55] Acknowledgements Recombinant V spinosum tyrosinase 6 7 8 9 10 11 12 13 The authors wish to thank Linda Fahrni for technical assistance and Dr Julian Ihssen and Dr Matthijs De Geus for critically reading the manuscript 14 References 1 Claus H & Decker H (2006) Bacterial tyrosinases Syst Appl... Identification of FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research Journal compilation ª 2010 FEBS M Fairhead and L Thony-Meyer ¨ 47 48 49 50 51 52 53 copper ligands in Aspergillus oryzae tyrosinase by sitedirected mutagenesis Biochem J 350 Pt 2, 537–545 Tatara Y, Namba T, Yamagata Y, Yoshida T, Uchida T & Ichishima E (2008) Acid activation of protyrosinase... oxidase from peach (Prunus persica L Cv Catherina) Molecular properties and kinetic characterization of soluble and membrane-bound forms J Agric Food Chem 55, 10446–10451 31 Gandia-Herrero F, Garcia-Carmona F & Escribano J (2004) Purification and characterization of a latent polyphenol oxidase from beet root (Beta vulgaris L.) J Agric Food Chem 52, 609–615 2094 M Fairhead and L Thony-Meyer ¨ 32 Tobias... proteins Biochem J 256, 1001–4 55 Pace C N & Scholtz J M (1997) Protein structure: a practical approach IRL Press, Oxford Supporting information The following supplementary material is available: Fig S1 DNA and amino acid sequence of V spinosum tyrosinase Fig S2 Calibration curve for size determination of selected tyrosinases Fig S3 Unfolding curves of selected tyrosinases Fig S4 Absorbance spectra of. .. (2000) Tyrosinase ⁄ catecholoxidase activity of hemocyanins: structural basis and molecular mechanism Trends Biochem Sci 25, 392– 397 45 Gerdemann C, Eicken C, Galla HJ & Krebs B (2002) Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure J Inorg Biochem 89, 155–158 46 Nakamura M, Nakajima T, Ohba Y, Yamauchi S, Lee BR & Ichishima E (2000) Identification... & Fuerst JA (2009) Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes BMC Microbiol 9, 5 20 Plonka PM & Grabacka M (2006) Melanin synthesis in microorganisms-biotechnological and medical aspects Acta Biochim Pol 53, 429–443 21 Steenbergen JN & Casadevall A (2003) The origin and maintenance of virulence for the human pathogenic... Trypsinization Trypsin digest of purified pro -tyrosinase was performed by dissolving 20 lg of proteomics grade TPCK treated porcine trypsin (Sigma-Aldrich) in 50 lL of 1 mm HCl and mixing it with an aliquot of pro -tyrosinase ( 10 mg), final volume 1 mL in 0.1 m Tris-HCl buffer (pH 8) The sample was then incubated at room temperature for up to 24 h Chemical denaturation of proteins Unfolding experiments using... of the bC-haemocyanin of Helix pomatia Eur J Biochem 248, 879–888 29 Espin JC & Wichers HJ (1999) Activation of a latent mushroom (Agaricus bisporus) tyrosinase isoform by sodium dodecyl sulfate (SDS) Kinetic properties of the SDS-activated isoform J Agric Food Chem 47, 3518–3525 30 Cabanes J, Escribano J, Gandia-Herrero F, GarciaCarmona F & Jimenez-Atienzar M (2007) Partial purification of latent polyphenol . Role of the C-terminal extension in a bacterial tyrosinase Michael Fairhead and Linda Tho ¨ ny-Meyer EMPA, Swiss Federal Laboratories for Materials. diphenolase activities in bacterial extracts (data not shown). Features of the amino acid sequence of the V. spinosum tyrosinase The amino acid sequence of the