Multifunctions of MelB, a Fungal Tyrosinase from Aspergillus oryzae

Abstract

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2)OH (reductant), the proenzyme bound dioxygen to give a stable (μ-η(2):η(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.

Controlling Dicopper Protein Functions

“…The fulllength melB pro-tyrosinase consists of 621 amino acid residues and most of the residues are well defined in the final electron density, except for some disordered loops in the flexible regions. Consistent with the homo-dimeric state at physiological condition found in our previous study, 35 very tight intermonomer interaction, which glued two monomers together through extensive hydrophobic and chargecharge interactions, have been observed. The monomer associates in a side-toside manner along a two-fold axis; thus, the substrate-binding pockets face alternate directions so as not to interfere with each other.…”

“…Consistent with the structural similarity, the holo-pro-tyrosinase has no tyrosinase activity, but the reduced pro-tyrosinase (deoxyform), produced by the reduction of the as-isolated enzyme with hydroxylamine (reductant), exhibited a reversible dioxygenbinding ability and the dissociation constant (K d ) was determined to be 52¯M. 35 As the K d value of molluscan hemocyanin was reported as 90¯M, 55 these affinity toward dioxygen is in a similar range. Leu2830 residue in the octopus hemocyanin Based on the structural similarity of the functional unit g of molluscan hemocyanin with melB pro-tyrosinase, we expected that the molluscan hemocyanin can get the phenolase and catecholase activities under some denaturation conditions.…”

“…35 The copper-binding domain of melB pro-tyrosinase is structurally similar to the active form of mushroom tyrosinase. 34,43 Therefore, this hydrolytic cleavage of the C-terminal domain induces little conformational change of the core domain, but simply causes the removal of the placeholder residue from the active site, retaining the homodimeric form as demonstrated by size-exclusion chromatography (Figure 4).…”

Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase

“…Tyrosinase-melB pro-form of tyrosinase was prepared by using pETH-melB plasmid as described previously (22,23). As for selenomethionine-substituted tyrosinase, expression plasmids were transformed into E. coli B834 (DE3) pLysS-competent cells.…”

“…The holo-pro-tyrosinase thus formed has no catalytic activity, but the trypsin treatment converts the holo-pro-form into the active form of the enzyme, in which the C-terminal domain (Gly 464 -Ala 616 ) is absent (cleaved) (23). Here, we successfully determined the crystal structures of the holo-pro-and the apo-pro-forms of melB tyrosinase from A. oryzae at a 1.39-and a 2.05-Å resolution, respectively, to provide the first detailed structural information about the fungal tyrosinase containing the C-terminal domain.…”