Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone

Abstract

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ∼392 residues and two L subunits of ∼150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ∼100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

Determination of tyrosinase substrate-binding modes reveals mechanistic differences between type-3 copper proteins

“…Tyrosine/L-Dopa ARTICLE tyrosine owing to the absence of a thioether bond 15 , and tyrosinase from A. bisporus performs hydroxylation despite the thioether bond because of the presence of V283 above the active site 8 . This provides a compelling and structurally founded explanation for the long-lasting debate on the different activities of type-3 copper proteins.…”

“…The catalytic mechanism of type-3 copper proteins has been studied extensively for the past two decades, recently assisted by the availability of three-dimensional structures [4][5][6][7][8] . Based on these structures, it is evident that the differences in function are due to variations in the residues surrounding the substrate-binding pocket.…”

The structure of the FnI-EGF-like tandem domain of coagulation factor XII solved using SIRAS

“…An advanced search for 'HO' as a ligand was performed using the PDBe web portal and resulted in nine hits [PDB entries 2y9x (2.78 Å resolution; Ismaya et al, 2011) Weis et al, 1991)]. The distances between Ho atoms and protein atoms were measured in Coot and the mean distance and the standard deviation were calculated.…”

Kinetic, Thermodynamic and Structural Studies of Native and N-Bromosuccinimide-Modified Mushroom Tyrosinase

“…In the sequence of MT, as shown in Figure 1, the residues numbered 61, 85, 94, 259, 263, and 296 are metal binding sites of Cu +2 which among, the amino acids numbered 61, 85, and 94 are binding sites of copper 1 (Cu +1 ) and amino acids numbered 259, 263, and 296 are binding sites of copper 2 (Cu +2 ). Also, the amino acids numbered 293-293 are cleavage sites (4,5). Four genes including Abppo1 (Uniprot protein sequence database entry Q00024) and Abppo2 (O42713) (6), and Abppo3 (C7FF04) and Abppo4 (C7FF05) (7) are responsible for coding of Agaricus bisporus tyrosinase.…”