Crystallization and preliminary X-ray crystallographic analysis of tyrosinase from the mushroom<i>Agaricus bisporus</i>

Ismaya, Wangsa T.; Rozeboom, H.J.; Schurink, Marloes; Boeriu, Carmen G.; Wichers, Harry J.; Dijkstra, Bauke W.

doi:10.1107/s174430911100738x

Cited by 32 publications

(28 citation statements)

References 26 publications

(27 reference statements)

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Emami¹,

Gheibi

2016

Biotech Health Sci

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Background: Mushroom tyrosinase (MT) as a metalloenzyme is a good model for mechanistic studies of melanogenesis. To recognize the mechanism of MT action, it is important to investigate its inhibition, activation, mutation, and modification properties. Objectives: In this study, the chemical modification of MT tryptophan residues was carried out by using N-bromosuccinimide (NBS) and then, the activity, stability, and structure of the native and modified enzymes were compared. Methods: Chemical modification of MT tryptophan residues was accomplished by enzyme incubation with different concentrations of NBS. The relative activity of native and modified MT was investigated through catecholase enzyme reaction in presence of dihydroxyphenylalanine (L-Dopa) as substrate. Thermodynamic parameters including standard Gibbs free energy change (∆G25°C) and Melting temperature (Tm) were obtained from thermal denaturation of the native and modified enzymes. The circular dichroism and intrinsic fluorescence techniques were used to study secondary and tertiary structure of MT, respectively. All experiments were conducted in 2015 in biophysical laboratory of Qazvin University of Medical Sciences and Islamic Azad University, Science and Research Branch, Tehran. Results: The relative activity reduced from 100% for native enzyme to 10%, 7.9%, and 6.4% for modified MT with different NBS of

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Section: Introductionmentioning

confidence: 99%

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

Emami¹,

Gheibi

2016

Biotech Health Sci

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“…Purification and crystallization of the enzyme were done as described elsewhere. 13 Briefly, tyrosinase crystals were obtained at room temperature using the hanging-drop vapor diffusion technique with drops made of a 1:1 mixture of protein solution (∼6 mg/mL) in 10 mM HEPES buffer, pH 7.5, and reservoir solution, containing 10% (w/v) PEG4000 in 100 mM sodium acetate buffer, pH 4.6, with 5 mM holmium chloride as additive. After 4À5 days two different crystal forms appeared in the drops, belonging to space groups P2 1 2 1 2 or P2 1 .…”

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confidence: 99%

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

Ismaya¹,

Rozeboom²,

et al. 2011

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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.

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“…Tyrosinase is associated with pigmentation catalyzing the first step in melanin biosynthesis – a pigment ubiquitously present in all phyla [6]. The structure of mushroom ( Agaricus bisporus ) tyrosinase (abTy) is a tetrameric protein with 2 heavy (H, 392 aa, ~43 kDa) and 2 light (L, 150 aa, ~14 kDa) chains (2.30 Å resolution, 2Y9W) [7, 8]. As a derivative of the ppo3 gene, the fungal enzyme is structurally homologous to other tyrosinases, only larger with 110 more residues present as loops connecting conserved secondary structural elements.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

Washington

Maxwell

Stevenson

et al. 2015

Archives of Biochemistry and Biophysics

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Tyrosinase(EC 1.14.18.1) catalyzes the monophenolase and diphenolase reaction associated with vertebrate pigmentation and fruit/vegetable browning. Tyrosinase is an oxygen-dependent, dicopper enzyme that has three states: Emet, Eoxy, and Edeoxy. The diphenolase activity can be carried out by both the met and the oxy states of the enzyme while neither mono- nor diphenolase activity results from the deoxy state. In this study, the oxidative cyclocondensation of 2-aminophenol (OAP) to the corresponding 2-aminophenoxazin-3-one (APX) by mushroom tyrosinase was investigated. Using a combination of various steady- and pre-steady state methodologies, we have investigated the kinetic and chemical mechanism of this reaction. The kcat for OAP is 75±2 sec−1, KMOAP=1.8±0.20.2emnormalmM, KMO2=25±40.2emnormalμitalicM with substrates binding in a steady-state preferred fashion. Stopped flow and global analysis support a model where OAP preferentially binds to the oxy form over the met (k7 ≫ k1). For the met form, His269 and His61 are the proposed bases, while the oxy form uses the copper-peroxide and His61 for the sequential deprotonation of anilinic and phenolic hydrogens. Solvent KIEs show proton transfer to be increasingly rate limiting for kcat/KMOAP as [O2] −>0 μM (1.38±0.06) decreasing to 0.83±0.03 as [O2]−>∞ reflecting a partially rate limiting μ-OH bond cleavage (Emet) and formation (Eoxy) following protonation in the transition state. The coupling and cyclization reactions of o-quinone imine and OAP pass through a phenyliminocyclohexadione intermediate to APX, forming at a rate of 6.91±0.03 μM−1s−1 and 2.59E-2±5.31E-4 s−1. Differences in reactivity attributed to the anilinic moiety of OAP with o-diphenols are discussed.

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Crystallization and preliminary X-ray crystallographic analysis of tyrosinase from the mushroomAgaricus bisporus

Cited by 32 publications

References 26 publications

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

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

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

Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

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