Heterologous expression and characterization of functional mushroom tyrosinase (AbPPO4)

Tyrosinases are an ubiquitous group of copper containing metalloenzymes that hydroxylate and oxidize phenolic molecules. In an application context the term ‘tyrosinase’ usually refers to ‘mushroom tyrosinase’ consisting of a mixture of isoenzymes and containing a number of enzymatic side-activities. We describe a protocol for the efficient heterologous production of tyrosinase 4 from Agaricus bisporus in Escherichia coli. Applying this procedure a pure preparation of a single isoform of latent tyrosinase can be achieved at a yield of 140 mg per liter of autoinducing culture medium. This recombinant protein possesses the same fold as the enzyme purified from the natural source as evidenced by single crystal X-ray diffraction. The latent enzyme can be activated by limited proteolysis with proteinase K which cleaves the polypeptide chain after K382, only one The latent enzyme can amino acid before the main in-vivo activation site. Latent tyrosinase can be used as obtained and enzymatic activity may be induced in the reaction mixture by the addition of an ionic detergent (e.g. 2 mM SDS). The proteolytically activated mushroom tyrosinase shows >50% of its maximal activity in the range of pH 5 to 10 and accepts a wide range of substrates including mono- and diphenols, flavonols and chalcones.

0.1 g l -1 BSA; pH 7.9 @ 25°C) for 16 h @ 16 °C followed by thermal denaturation of the enzymes by heating to 65 °C for 10 min. Before transformation the ligation mixture was incubated with an additional 10 units of SmaI for 2 h @ 25 °C in order to suppress background colonies arising from transformation of religated vector.
Chemically competent cells (E. coli NEB 5-alpha) were prepared according to the CaCl 2 -method 4 and stored frozen at -80 °C. 6 µl of the ligation mixture (after additional digestion with SmaI) were transformed into NEB 5-alpha cells using the heat shock protocol. 5 Transformants were selected on LB agar plates with 100 mg l -1 Na-Ampicillin by incubation at 37 °C overnight. The colonies that formed on this selective media were tested for the presence of the AbPPO4-gene in the right orientation by colony PCR 6  pH 8.6 @ 25°C), ddH 2 O to 10 µl and a few nl from the bacterial colony to be tested.
PCR was carried out as follows: 7 min @ 95 °C for cell lysis and initial denaturation of the DNA, 35 cycles of 40 s @ 94 °C, 60 s @ 59 °C, 140 s @ 72 °C, where the annealing temperature (59 °C) was initially set to 67 °C and was lowered by 1 °C per cycle for the first 9 cycles, and 10 min @ 72 °C for the final elongation. Positive clones were taken into liquid culture (12 ml SB with 100 mg l -1 Na-Ampicillin, incubation overnight at 37 °C and 250 min -1 ) which were subsequently used for plasmid DNA extraction (Wizard ® Plus SV Minipreps DNA Purification System, Promega). The purified plasmids were analyzed in the region of the insert by Sanger sequencing of both DNA strands (Microsynth; Vienna, Austria).
In order to bring the AbPPO4-gene in frame with the fusion partner glutathione Stransferase (GST) encoded by the pGEX vector a single deoxyadenosine monophosphate was inserted in the region before the open reading frame of the gene for AbPPO4. This was done applying the Q5 ® Site-Directed Mutagenesis Kit (NEB) according to the manufacturers recommendations with the two primers pGEX-6P-1:2_AbPPO4_fwd and pGEX-6P-1:2_rev resulting in a primer annealing temperature of 72 °C. The resulting DNA constructs were transformed into E. coli NEB 5-alpha and analyzed by colony PCR and Sanger sequencing as described for the initial cloning step.
chemically competent E. coli BL21(DE3). The resulting transformants were selected on LB with 100 mg l -1 Na-Ampicillin and positive transformants were tested for the presence of the expression vector by colony PCR as described for the cloning of the gene for AbPPO4. One transformant per variant of the expression plasmid was grown in 10 ml of liquid medium (LB with 2 mM MgSO 4 and 100 mg l -1 Na-Ampicillin) overnight @ 37 °C and 250 min -1 . These cultures were then concentrated to 1 ml by centrifugation at 1000 x g for 2 min @ 30 °C, mixed with an equal volume of 50 % (v/v) glycerol and snap-frozen in liquid nitrogen. Those cryostocks were stored at -80 °C and served as an invariant source of inoculum for the subsequent expression cultures.
Auto-inducing medium was applied for the expression, namely ZYM-5052 7 without the trace element solution and with added Cu 2+ to increase the activity of the heterologous enzyme. 8  Protein crystallography: model building. Initial phases were obtained by molecular replacement using PHASER 11 supplied with structure factors derived from the crystal structure of AbPPO4 purified from the natural source (PDB 4OUA, chain B). 12 For solving the structure programs from both the CCP4 13 and PHENIX 14 suites were applied. Refinement and rebuilding was performed by employing PHENIX (phenix.refine) and Coot. 15 In order to improve the model and reduce model bias, a first refinement step was carried out in which the atoms were "shaken" by displacing them using a r.m.s.d. distance of 0.8 Å. 16 The resulting partially randomized model was then subjected to simulated annealing using torsion angle dynamic to minimize model bias as much as possible.
Synthesis of tyrosine methyl esters. L-tyrosine (0.9 g, 5 mmol) was dissolved in methanol and thionyl chloride (0.545 ml, 7.5 mmol) was added slowly at 0 °C. After refluxing for 2 h, the reaction mixture was poured into ice water (20 ml), neutralized with NaHCO 3 and extracted with EtOAc (2 x 20 ml). The organic extracts were        The reaction rates in buffers with pH > 7 were scaled by the relative reaction rate in 50 mM of the respective buffer substance (pH ≤ 7: Na-citrate, pH 8 -8.8: Tris-HCl, pH 9.5 -10.5: Glycin/NaOH) set to pH 7 and 50 mM sodium citrate buffer at pH 7 as the reference system.
Reaction rates without enzyme at the respective pH were subtracted from each data point, but the autooxidation rates of tyrosine were not significantly different from zero even at pH 10.5. One data point represents the average of three measurements, the error bars show ± one standard deviation.   range tested). Blue and grey diamonds represent measured slopes, data point marked with grey diamonds indicate a significant contribution of substrate inhibition. Those data points were excluded from the data analysis yielding the least Michaelis-Menten models. Reaction rates predicted using those models are shown as red curves. Table 2 Tyrosine  Figure S8: Structures of substrates accepted by activated AbPPO4