A Novel Tyrosinase from Armillaria ostoyae with Comparable Monophenolase and Diphenolase Activities Suffers Substrate Inhibition

Abstract

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyae strain C18/9 (AoTyr) was characterized, and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the activities. In silico analysis and mutagenesis experiments showed that residues 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the replacement of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (k_cat/K_m ratios) (1.63-fold) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. IMPORTANCE Tyrosinase is able to oxidize various phenolic compounds, and its ability to convert monophenols into diphenols has caught great attention in the research field and industrial applications. However, the utilization of tyrosinase for the industrial synthesis of catechols has been limited due to the fact that the monophenolase activity of most of the known tyrosinases is much lower than the diphenolase activity. In the present study, a novel tyrosinase with comparable monophenolase and diphenolase activities was characterized. The enzyme exhibits substrate inhibition in both monophenolase and diphenolase activities. In silico analysis followed by mutagenesis experiments confirmed the important roles of residues 262 and 266 in the substrate inhibition and activity modulation of the enzyme, and the D262N variant showed an enhanced monophenolase/diphenolase catalytic efficiency ratio compared to the wild-type enzyme.

Keywords: Armillaria ostoyae; diphenolase; monophenolase; mutagenesis; substrate inhibition; tyrosinase.

FIG 1

Multiple-sequence alignments between AoTyr and highly homologous tyrosinases from A. ostoyae (GenBank accession number SJL09928.1), L. edodes (UniProt accession number Q96TI3), P. arcularius (UniProt accession number Q65Z70), P. nameko (UniProt accession number A7BHQ9), and A. bisporus (UniProt accession number C7FF05). Histidine residues participating in copper coordination are marked by blue stars, and conserved regions such as the type 3 copper-binding motif, the Y-X-Y motif, the YG motif, and the C-X-X-C motif are underlined.

FIG 2

Structure analysis of AoTyr. (A) Modeled latent AoTyr structure. The N-terminal (N′) domain is in blue, the C-terminal (C′) domain is marked in magenta, and the histidine residues involved in type 3 copper binding are depicted in orange. (B and C) View of the active site of the N-terminal domain. An l-tyrosine molecule (in cyan) is docked in the active center. Residues involved in copper coordination are colored and labeled in orange, and residues D262 and D266 are labeled and colored in magenta.

FIG 3

(A) SDS-PAGE analysis of AoTyr after Ni affinity chromatography. Lanes: M, molecular weight marker; 1, cell before induction; 2, cell after induction; 3, latent AoTyr purified by Ni affinity chromatography (eluted with 200 mM imidazole in buffer); 4, activated AoTyr (activation with trypsin). The weights of the used standard protein are indicated at the left of the gel. Protyrosinase (66 kDa) in lane 3 and tyrosinase (46 kDa) in lane 4 are indicated with red arrows. (B) SDS activation of latent AoTyr. For the control, the same amount of water was used as SDS. (C) pH and temperature profile of activated AoTyr. The substrate used in both pH and temperature profile assays was l-DOPA at a final concentration of 1 mM.

FIG 4

Effects of organic solvents (A), metal ions, and other compounds (B) on AoTyr activity. In the reaction mixtures, 1 mM l-DOPA was used as the substrate. For the control, the same amount of water was used as the chemicals or agents.

FIG 5

Substrate specificity of trypsin-activated AoTyr toward different monophenols. Product analysis was performed using HPLC at 270 nm. The red lines represent substrates before the catalytic reaction, and the blue lines represent the corresponding products. (A) Phenol. (B) 2,4-Dimethylphenol. The AUCs of the two major peaks are 608 and 676, respectively. (C) 3,4-Dimethylphenol. Docking experiments indicated that the C-6 atom of the compound will be hydroxylated by the enzyme. (D) 2,3,5-Trimethylphenol. The AUCs of the two major peaks are 582 and 262, respectively.

FIG 6

Substrate inhibition fitting curve of AoTyr (A) and the D262N (B), D266N (C), and D262N/D266N (D) variants on l-tyrosine and l-DOPA.