Activation Mechanism of the Streptomyces Tyrosinase Assisted by the Caddie Protein

Abstract

Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated as a complex with a "caddie" protein that assists the transport of two copper ions into the active center. In this complex, the Tyr residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of μ-η:η-peroxo-dicopper(II) and Cu(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic conditions and Fourier transform infrared spectroscopic analysis using CO as a structural probe under anaerobic conditions indicated that the copper transportation process to the active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the caddie protein, which is triggered by the conversion of the Tyr residue to dopaquinone, may ensure the generation of fully activated tyrosinase.

Catalytic mechanism of the tyrosinase reaction toward the Tyr98 residue in the caddie protein

“…The newly added oxygen (Oζ2) is within the coordination bond distance from Cu A-3 and near to Cu B-2 (S1E Fig). The complex between Cu A-3 and the oxygenated Tyr 98 may correspond with the Cu(II)-bound dopasemiquinone observed in the solution state [29]. Specifically, Cu A-3 is in a bipyramidal trigonal coordination cage, in which the axial ligands are the Oζ2 atom added to the caddie Tyr 98 and the Nε atom of His 38 , and equatorial ligands are the Oη atom of the caddie Tyr 98 , the Nε atom of His 54 , and the bridging oxygen atom at the Wat 3 site.…”

“…In the previous study [29], we demonstrated that the addition of NH 2 OH stimulates the caddie proteins to aggregate, resulting in the release of tyrosinase from the complex. The aggregation is likely triggered by the formation of reactive dopaquinone on the caddie Tyr 98 residue.…”

“…After the crystal structure of tyrosinase was shown by our group [12], its catalytic mechanism has been actively discussed by other groups [4,6,15,23,24,27,28,30–33,40–42]. Since tyrosinase can react with the Tyr 98 residue of the caddie protein [29], the Tyr 98 residue is expected to adopt a similar binding position of L -tyrosine as a genuine substrate of tyrosinase. However, free L -tyrosine, which lacks the structural restraints as compared with the Tyr 98 residue as a part of the caddie protein, may be bound deeply into the active-site pocket.…”

“…The binding sites for the additional copper ions (Cu C , Cu D , and Cu E ) in the caddie protein and the hydrogen-bonding network around the tyrosinase active site were found to be important for the effective transfer of Cu(II). Our group has recently demonstrated that the incorporation of copper ions into tyrosinase and the following release of copper-bound tyrosinase progress more quickly in the presence of NH 2 OH, which can reduce the met form to the deoxy form, but not the oxy form, under aerobic conditions [29]. Cu(I), but not Cu(II), must be suitable species to be incorporated into the active center of tyrosinase.…”

“…The dopaquinone must be formed as a result of the catalytic activity of the oxy-tyrosinase. The ultraviolet-visible (UV-vis) and resonance Raman spectroscopic analyses indicated that the Tyr 98 residue is converted to dopaquinone through the formations of μ-η 2 :η 2 -peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates [29], although the formation of dopaquinone is a speculation from the fact that the modified caddie is easily aggregated. Reaction intermediates were able to be trapped under the conditions at which the aggregation of the caddie was inhibited.…”