Crystallization and preliminary X-ray crystallographic analysis of latent isoform PPO4 mushroom (Agaricus bisporus) tyrosinase

Abstract

Tyrosinase exhibits catalytic activity for the ortho-hydroxylation of monophenols to diphenols as well as their subsequent oxidation to quinones. Owing to polymerization of these quinones, brown-coloured high-molecular-weight compounds called melanins are generated. The latent precursor form of polyphenol oxidase 4, one of the six tyrosinase isoforms from Agaricus bisporus, was purified to homogeneity and crystallized. The obtained crystals belonged to space group C121 (two molecules per asymmetric unit) and diffracted to 2.78 Å resolution. The protein only formed crystals under low-salt conditions using the 6-tungstotellurate(VI) salt Na6[TeW6O24] · 22H2O as a co-crystallization agent.

Keywords: Agaricus bisporus; polyoxometalates; polyphenol oxidase 4; tyrosinases; zymogens.

Figure 1

Schematic illustration of the polypeptide chain of PPO4 mushroom tyrosinase. The polypeptide chain of active tyrosinase (core region) is coloured red. The C-terminal domain is coloured orange. The missing C-terminal tail is coloured purple. Aa, amino acids; A-TYR, active tyrosinase; L-TYR, latent tyrosinase.

Figure 2

Crystal images of PPO4 mushroom tyrosinase (left, total drop image; right, enlarged image). (a) Wispy microcrystals (sea urchins) obtained using MgCl₂ as a crystallization additive (10% PEG 4000, 15 mM MgCl₂, 25 mM Tris–HCl pH 7.5). (b) Flat rod-shaped crystals obtained using the POM as a crystallization additive [10% PEG 4000, 1 mM Na₆[TeW₆O₂₄]·22H₂O, 25 mM Tris–HCl pH 7.5].