Figure 1.
Recombinant protein hTyrCtr consists of the intra-melanosomal portion of human tyrosinase.
A: The domain structure of human tyrosinase was obtained using the SMART (http://smart.embl-heidelberg.de). Red = signal peptide; green = EGF-like domain; gray = catalytic (tyrosinase) domain; blue = trans-membrane domain. B: The alignment of human tyrosinase and human tyrosinase intra-melanosomal domain sequences. The intra-melanosomal domain is located between the N- terminal signal peptide and a truncated C-terminus. The 18 N- and 70- C-terminal residues of the boundary are shown in red. A TEV cleavage site and a 6His-Tag are shown in green. Potential N-glycosylation sites, N86, N111, N161, N230, N290, N337, and N371, and copper-binding sites coordinated by His residues H180, H202, and H211 (A-site) and H363, H367, and H390 (B-site), are shown in magenta and blue, respectively. Locations of two temperature-sensitive mutations R422Q/W are shown. The sequence molecular mass of hTyrCtr is 53.129 kDa and the calculated pI is 5.70. Sequence fragments related to protein domains are shown by background colors: light green (EGF-like); light grey (tyrosinase) and light blue (trans-membrane helix).
Figure 2.
Purification and characterization of hTyrCtr.
A: IMAC using a HisTrap 5 ml column. The arrow indicates the start of the imidazole gradient. B: Gel filtration using Superdex 75 16/60 HR. Chromatography profile monitored at 260 nm (purple lines) and 280 nm (green lines). The inserts show the diphenol oxidase activity of hTyrCtr measured in separate tube for each fraction after 30 min of incubation at 37°C with 3 mM L-DOPA in 50 mM sodium phosphate buffer, pH 7.5. C: SDS-PAGE (top panel) and Western blot (bottom panel) showing stepwise purification of hTyrCtr. From left to right: L, protein ladder; 1, total lysate of larvae expressing hTyrCtr; 2, flow through; 3, sample after 5 ml HisTrap; 4, sample after Superdex 75. D: Sedimentation equilibrium of hTyrCtr. The protein concentration gradient (280 nm) versus radial distance is indicated. The red line shows calculated fit for an ideal monomer and blue circles the experimental values. The top panel shows the residuals of a fitted curve to the data points. Calculated fit of monomer was obtained assuming that the average partial specific volume of glycans is 0.63 cc/g and that the protein contains 10% carbohydrate.
Figure 3.
Gel filtration and enzymatic activities for the pure recombinant hTyrCtr and two temperature sensitive mutant variants.
Chromatographies were using Superdex 75 10/30 HR: hTyrCtr (B) and of R422Q and R422W (E). The elution points of molecular mass standards are shown at the top for reference. Panels A, C and D show test tubes containing the L-DOPA colorimetric reactions for each protein fraction of hTyrCtr, R422Q and R422W, respectively. Brown color (intensity proportional) in tube indicates diphenol oxidase activity. Corresponding Western blots bands were labeled by horizontal arrows.
Table 1.
Purification of recombinant hTyrCtr and two temperature sensitive variants from 10 g of larval biomassa.
Figure 4.
Kinetic analysis of hTyrCtr and two mutants.
Michaelis-Menten plots of monophenolase (A, B) and diphenol oxidase (C, D) activities of hTyrCtr (blue) and two mutants, R422Q (red) and R422W (green), as a function of L-tyrosine and L-DOPA concentrations. Enzyme assays were measured at 37°C (A, C) and 31°C (B, D). The lines represent nonlinear fits to the Michaelis-Menten equation. Experiments were performed in triplicate and error bars represent standard deviations.
Table 2.
Kinetic parameters for tyrosinase-catalyzed reactions.
Table 3.
Effect of Inhibitors on enzymatic activity of hTyrCtr is shown by IC50 values.
Figure 5.
N-glycosylation sites determined by MS are mapped to the human tyrosinase protein structure modeled as described in methods section. The protein backbone structure is shown by magenta ribbon. Two copper atoms, CuA and CuB, which coordinated by His residues are shown in orange. Fully and partially occupied N-glycosylation sites are represented by red and yellow spheres, respectively. Two potential N-glycosylation sites, not determined in in present study, are shown in grey. The location of mutant variants is indicated in green.
Figure 6.
Catalytic efficiencies of hTyrCtr and mutants R422Q and R422W as a function of temperature.
Panel A shows a schematic view of first two steps of the melanin biosynthesis pathway regulated by tyrosinase. The catalytic efficiency (kcat/Km, mM−1 min−1) of the monophenolase (B) and diphenol oxidase (C) activity obtained from the Table 2 for hTyrCtr and mutant variants R422Q and R422W at 37 and 31°C are shown by light blue and dark blue bars, respectively; *p<0.05. Protein structure of the wild type hTyrCtr and temperature-sensitive mutant variants, R422Q and R422W are shown on Panels D, E and F, respectively. Structural superposition of human tyrosinase hTyrCtr and the bacterial tyrosinase (PDB file: 3nm8) shown by grey and cyan, respectively. Temperature-sensitive mutations at positions 402, 406 and 422 are located in the same structural fragment shown by orange.
Figure 7.
Far-UV CD Spectra of hTyrCtr and temperature sensitive mutants R422Q/W.
CD spectra for hTyrCtr and two mutants, R422Q and R422W, are shown by blue, red, and green solid lines, respectively. Dashed lines show correspondent spectra measured in the presence of 0.5 mM tyrosine. Measurements were performed at 37°C (A) and 31°C (B). Scans (190–260 nm) were performed in 50 mM sodium phosphate buffer, pH 7.5 at protein concentrations of 0.2 mg/ml. Inserts: spectral differences at the range 200–230 nm are shown. Histograms C and D show ellipticity (Θ) ratios (%) in the absence (C) or the presence (D) of tyrosine with data from spectra shown in Panels A, B. The ratios were calculated as 100% × (Θ) 31°C/(Θ) 37°C determined at fixed wavelengths of 208, 222 nm (α-helix), and 215 nm (β-sheet). Dashes show a 100% level.