Importance of Copper Tyrosinase Binding for Catalytic Activity

Abstract

Tyrosinase is a copper-containing enzyme responsible for melanin pigment biosynthesis. However, the detailed structure of human tyrosinase has not yet been resolved, along with the identification of the key sites for its catalytic activity. We used site-directed mutagenesis to identify the critical residues for the copper binding of human tyrosinase. Seven histidine mutants (H180A, H202A, H211A, H363A, H367A, H389A, and H390A) in the two copper-binding sites were generated by substituting histidine for alanine, and catalytic activities toward hydroxylation and oxidation reactions were characterized. The tyrosine hydroxylase activities of the mutants at the CuA site were approximately 50% lower than those of the wild-type enzyme, while the dopa oxidation activities were similar to those of the wild-type enzyme. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylase and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. Furthermore, the H363, H367, and H390 residues appear to be directly involved in copper-binding. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetics and pharmaceutical industries.

Significance

We demonstrate that six histidine residues of tyrosinase play key roles in its hydroxylation and oxidation activities, at least partially. H180A, H202A, and H211A mutants at the CuA-binding site significantly abolished tyrosine hydroxylation activity but not dopa oxidation activities, whereas the H363A, H367A, and H390A mutants at the CuB-binding site lacked both activities, supporting their essential roles for catalytic activity. Given the main function of tyrosinase in melanin production, this information will be of great value in further predicting the structure and designing new inhibitors for the treatment of pigment-associated conditions or for cosmetics development.

Introduction

Melanin biosynthesis is a complex pathway involving enzymatic and chemical reactions, which is restricted to the melanocytes in mammals. Tyrosinase (monophenol monooxygenase, EC 1.14.18.1) is a key enzyme in the melanin synthesis pathway. Moreover, tyrosinase is the only human melanogenic enzyme with well-established in vivo catalytic activity, catalyzing several steps in melanin synthesis, including the hydroxylation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-dopa) and its subsequent oxidation to dopaquinone. The mature human tyrosinase (529 amino acids long) includes an 18-amino acid N-terminal signal peptide that targets the nascent polypeptide to the endoplasmic reticulum for folding, modification, and sorting. Tyrosinase is a copper-containing metalloprotein belonging to the type-3 copper protein family, together with catechol oxidases and hemocyanins. These proteins are abundant in mammals, plants, fungi, and bacteria, and the active sites are well conserved among the different species. By synthesizing melanin, tyrosinase has a protective effect against ultraviolet radiation-induced damage, but can also cause hyperpigmentation leading to esthetic problems and melanoma. Moreover, the absence of tyrosinase activity is associated with oculocutaneous albinism in many animal species, including humans. As such, human tyrosinase is a quite attractive target for medical and industrial applications. In particular, the development and screening of potent inhibitors of tyrosinase has received substantial attention in the cosmetic industry.

To date, two crystal structures of catechol oxidase, three crystal structures of hemocyanin, and two crystal structures of tyrosinases from Streptomyces castaneoglobisporus and Bacillus megaterum have been resolved. Unfortunately, there is still no crystal structure of human tyrosinase; however, a reliable model could be generated on the basis of the amino acid sequence and active sites. Six histidine residues, which are provided by a four-helical bundle, coordinate the two copper ions (CuA and CuB) in the active site. Tyrosinase contains seven N-glycosylation sites, two putative copper-binding sites (CuA and CuB), and one transmembrane domain, followed by a relatively short carboxyl tail that contains the essential signals for sorting and targeting to the melanosomes. However, there is no obvious evidence for the direct binding of human tyrosinase to copper. A comparison study of the amino acid sequences of tyrosinases, catechol oxidases, and hemocyanins suggested that the two homologous regions CuA and CuB may be involved in the binding of copper atoms, which are necessary for its catalytic activity. Mutational analysis of human tyrosinase also suggested that the CuA and CuB sites are both required for copper binding and catalytic activity.

In the present study, we sought to demonstrate if the seven copper-coordinating histidine residues (H180, H202, H211, H363, H367, H389, and H390) around the CuA and CuB sites are necessary for tyrosinase catalytic activities and structure folding. Nakamura et al. previously reported the seven essential histidine residues, H63, H84, H93, H290, H294, H332, and H333, for tyrosinase activity of Aspergillus oryzae, and demonstrated that replacement of each residue with asparagine resulted in mutant enzymes exhibiting no activities. Moreover, a crystallographic analysis of Palinurus interruptus hemocyanin showed that one of the pairs of copper ions, CuA, is surrounded by residues H196, H200, and H226, while the other, CuB, is surrounded by residues H346, H350, and H386. Based on this background, in the present study, the mutation positions were selected by sequence comparison of the tyrosinase from H. sapiens with other tyrosinases from Mus musculus, Oryzias latipes, Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lavendulae, Streptomyces lincolnensis, Neurospora crassa, A. oryzae, and Sinorhizobium meliloti, and focused at positions of seven histidine residues (H180, H202, H211, H363, H367, H389, and H390). Toward this end, we mutated each histidine residue to alanine, as a non-polar amino acid with a neutral small side chain, using a site-directed mutagenesis approach. We then compared the hydroxylation and oxidation activities of these mutants. These findings can provide insight into the essential residues responsible for the catalytic properties of human tyrosinase, which will be of great value in predicting the structure and designing new inhibitors.

Materials and Methods

Cloning and site-directed mutagenesis of the human tyrosinase gene

We previously reported the cloning of the human tyrosinase gene into the pHis vector, along with protein expression and purification. To improve these last two steps, in this study, we used a PCR approach to reclone the gene into the pET-26b(+) expression vector, which contains a 6x His-tag at the C-terminal, using Nde I and Xho I (Takara Shuzo, Otsu, Shiga, Japan) as restriction sites, including the stop codon.

Site-directed mutagenesis to replace the six His residues with Ala was performed using Quick Change Site-Directed Mutagenesis Kit according to the manufacturer’s manual.

Primers (COSMO Genetech, Seoul, Korea) used for cloning and mutation generation are listed in Table 1. Plasmids were transformed into XL-10 competent cells and were sequenced by a commercial supplier (Bionics, Seoul, Korea). Plasmids confirmed by sequencing were transformed into E. coli strain BL21 Star (DE3) (Novagene) for protein expression.

Expression of recombinant tyrosinase in E. coli

Production of the recombinant wild-type tyrosinase and mutant enzymes was performed by inoculating E. coli strain BL21 with each construct, followed by overnight culture at 37C in Luria-Bertani culture medium supplemented with 30 μg/mL kanamycin and 1.0 mM CuSO4, and then induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich) at an optical density at 600 nm of 0.3–0.4 for 12 h. The induced cells were harvested by centrifugation at 10,000 ×g for 10 min at 4C, washed three times in potassium phosphate buffer (pH 6.8), and resuspended in 10 ml of Buffer A containing 5.0 mM EDTA, 1.0 mM CuSO4, and 100 μM phenylmethanesulfonyl fluoride. The cells were then subjected to sonication using an ultrasonic processor for 20 min at 30–40 W with 9-s pulse on and 1-s pulse off. After centrifuging the lysate at 20,000 ×g for 10 min, the supernatant was collected and stored at 4C until analysis.

Purification of recombinant tyrosinase in E. coli

The His-tagged wild-type tyrosinase and mutant enzymes were purified by applying the lysate to a diethyaminoethyl (DEAE)-Sephacel column, following immobilization in a metal-affinity column. The unbound protein fractions after passing through the DEAE-Sephacel column were subjected to affinity column chromatography with Ni-conjugated agarose (Novagen). The column was washed with 50 mM potassium phosphate buffer:500 mM NaCl:1% Triton X-100, pH 7.8 (Buffer B) containing 10 mM imidazole, and the protein was eluted with Buffer B containing 100 mM imidazole followed by dialysis against Buffer A to remove the imidazole. The dialyzed purified wild-type tyrosinase and mutant enzymes were then used for the subsequent experiments. Unless otherwise indicated, all purification procedures were performed either at 4°C or on ice.

Enzyme assay and kinetics analysis

The tyrosine hydroxylase activity of wild-type and mutant human tyrosinase was measured as described by Tripathi, Chaya Devi, & Ramaiah. The dopa oxidase activity of tyrosinase was determined at 37°C by spectrophotometric monitoring at 475 nm for generation of the dopachrome product of the reaction as described by Hearing et al. In brief, the reaction mixtures contained 3.0 mM l-3,4-dihydroxyphenylalanine (l -dopa, dopachrome = 3600 M-1cm-1; Sigma-Aldrich), 50.0 mM Tris-HCl buffer (pH 7.5), and 0.05 mL of enzyme solution in a total volume of 1.2 mL. The steady-state rate was defined as the slope of the linear zone of absorbance-versus-time curves. Non-enzymatic reaction rates served as controls and were subtracted from the reaction rates determined in the presence of the enzyme. One unit of enzyme activity was defined as 1.0 mol of product formed per minute under the above experimental conditions. The specific activity was defined as units of enzyme activity per milligram of protein. To determine the substrate specificity, various mono-, di-, and tri-hydroxyphenol compounds were used as substrates. The rate of the reaction for these different substrates was measured at the maximum absorption of the reaction product between 300 and 500 nm. Relative activity was determined from the amount of enzyme that caused a change in absorbance of 0.001/min and was expressed with respect to the activity of l -dopa. Kinetic parameters for l-tyrosine and l -dopa were determined by the Lineweaver-Burk plot method. The parameters (with standard deviation) were determined in five separate experiments. Protein concentration was determined by the bicinchoninic acid assay reagent using bovine serum albumin as the standard protein.

Electrophoresis

Denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was carried out according to the method of Laemmli on 12.5% gels. The molecular-mass markers were SDS molecular weight standard markers (Bio-Rad) containing phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Coomassie Blue R-250 was used for protein staining.

Three-dimensional structure modeling of tyrosinase

Structural modeling of tyrosinase was performed by ‘SWISS-MODEL EXPASY’ software provided by Biozentrum, the center for molecular life sciences in Basel University (Basel, France). Modulation of predicted protein models was carried out with ‘Autodock tools’ software provided by the Scripps Research Institute (California, USA). Visualization of protein models were performed by ‘UCSF Chimera package’ from the Resource for Biocomputing, Visualization and Informatics (RBVI) in the University of California (California, USA),

Results

Expression and purification of the recombinant wild-type tyrosinase and mutants

Compared to our previous expression plasmid pHis-Tyrosinase containing the entire sequence for human tyrosinase and an N-terminal polyhistidine sequence tag, recloning of the tyrosinase gene into the pET26b(+) expression plasmid containing a C-terminal 6x His-tag successfully enhanced enzyme expression and catalytic activity. The recombinant pET26b(+)- human tyrosinase (pET-hTyr) could produce large amounts of pure and active tyrosinase (66 kDa) in the bacterial expression system. A high yield (approximately 34%, data not shown) of tyrosinase expression was obtained using E. coli BL21 Star (DE3) cells followed by purification, which was greater than that obtained in our previous study (approximately 19%), demonstrating the efficiency of the 6x His tag at the C-terminal rather than N-terminal. The overall purification was estimated to be increased by about 49-fold, and approximately 2.75 mg/L of purified tyrosinase was obtained. The purified recombinant human tyrosinase appeared as a single protein band corresponding to a molecular weight of 66,000 on the SDS-PAGE gel, which was used for subsequent characterization.

All of the tyrosinase mutants (H180, H202, H211, H363, H367, H389, and H390) were expressed and purified under identical conditions, and appeared as single protein bands on the SDS-PAGE gel. All mutants showed acceptable soluble, expressed, and purified enzyme concentrations in comparison with the wild-type tyrosinase.

Activity of the recombinant wild-type tyrosinase and mutants

The wild-type tyrosinase was expressed as a soluble and functional enzyme, showing clear hydroxylation and oxidation activities, and all six mutants showed lower activities compared to those of the wild-type. The majority of the mutant enzymes, except for the H389A mutant, showed significantly lower tyrosine hydroxylase (monophenolase) activity for the l-tyrosine substrate, indicating that these mutated residues are essential for tyrosine hydroxylation activity, whereas dopa oxidase (diphenolase) activity was reduced significantly in four mutants (H363A, H367A, H389A, and H390A) around the CuB site. Three mutants (H180A, H202A, and H211A) around the CuA site retained partial activity up to 88%, 76%, and 89%, respectively, for the dopa oxidation reaction compared to that of wild-type tyrosinase. This finding suggested that the copper binding of three histidine residues around CuA is essential for the aromatic substrate l-tyrosine. Moreover, structure modeling for human tyrosinase using Swiss-model Expasy software based on tyrosinase-related protein 1 structure, which is already resolved with X-ray crystallography, predicted that these three residues are also involved in direct binding to the copper atom.

Kinetic properties of the recombinant wild-type tyrosinase and mutants

Kinetic parameters of the wild-type human tyrosinase and mutants were determined by the Lineweaver–Burk plot and are shown in Table 3. The Km values of the mutant enzymes for the l-tyrosine hydroxylation reaction were similar to those of the wild-type enzyme. This result indicated that histidine residues around both the CuA- and CuB-binding sites are not involved in l-tyrosine substrate binding. By contrast, the Km values of the mutant enzymes for the l-dopa oxidation reaction were more variable. The three mutants around the CuA site showed similar Km values to those of the wild-type enzyme, but Km values of the H363A and H367A mutants were nine-times lower than those of the wild-type enzyme, suggesting that these two histidine residues may affect the binding affinity with the l-dopa substrate as well as copper binding. Furthermore, according to the kcat values for the wild-type tyrosinase and mutants, the three histidines bound to the CuA site are more critical for the l-tyrosine hydroxylation reaction, whereas the histidine residues at the CuB site play a more essential role for the l-dopa oxidation reaction.

In the predicted 3D tyrosinase model (Figure 3), the most likely binding sites of tyrosinase for CuA were determined to be H180, H202, and H211, which bind with copper atoms directly. Similarly, at the CuB site, the copper atom was coordinated by three histidine residues, H363, H367, and H390, through direct binding. H389 could be involved in catalytic reactions with indirect cooperation at the CuB region in human tyrosinase.

Discussion

Previous studies of tyrosinase showed that histidine residues are critical for its catalytic activity. In A. oryzae tyrosinase, the substitution of three copper-coordinating histidine residues (H63, H84, and H93) with Asparagine at the CuA site largely decreased copper binding by approximately 50%, suggesting that these residues are essential for copper binding. Moreover, these residues were deemed to be critical for catalysis given the decrease of the copper content to one copper atom and the loss of mono- and diphenolase activities with l-tyrosine and l-dopa, respectively (Nakamura et al., 2000). Similarly, the decrease of copper content in the histidine mutants (H93A, H116A, and H125A) of a polyphenol oxidase, type-3 copper enzyme of C. grandiflora confirmed that these residues are essential for copper binding. (Kaintz, Mayer, Jirsa, Halbwirth, & Rompel, 2015). Consistent with these previous reports, in this study, we confirmed that three histidine residues, H180, H202, and H211, of human tyrosinase around the CuA site are essential for enzyme activity as determined by site-directed mutagenesis. These three histidine residues may be directly involved in copper binding to catalyze the l-tyrosine hydroxylation reaction.

H290, H284, H332, and H333 around CuB site were found to be essential residues for catalytic activity and to act as copper ligands of the activated tyrosinase from A. oryzae (Nakamura et al., 2000). Spritz et al. (Spritz et al., 1997) described that human tyrosinase contains four conserved histidine residues (H363, H367, H389, and H390). They further showed that the H390A mutant abolished catalytic activity but did not decrease copper binding, whereas the H389 substitution resulted in complete lack of activity, suggesting that only H389 has a critical role in copper binding for human tyrosinase, despite the recognition of H390 as the conserved residue in all putative copper binding positions (Spritz et al., 1997). However, we found that the H363, H367, and H390 residues of human tyrosinase are likely directly involved in copper binding based on evaluations of catalytic activity and structure modeling. Furthermore, in contrast to these previous findings, H389 did not abolish the catalytic activity for l-tyrosine hydroxylation and showed indirect binding with the copper atom at the CuB site. It may need further study to elucidate the role of each histidine residue for catalytic activity.

Thus, we conclude that one pair of copper ions, CuA, directly binds to three histidine residues (H180, H202, and H211), while the other, CuB, directly binds to three other histidine residues (H363, H367, and H390) and indirectly binds to H389. We further assume that copper binding at one site may facilitate copper binding at the other site. However, the crystal structure of human tyrosine is required to identify the crucial residues for copper binding in catalyzing the reactions.

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