Characterization of Tyrosinase Enzyme From Native Bacillus Megaterium Sp. Strain M36

Valipour, Ebrahim; Arikan, Burhan

doi:10.15414/jmbfs.2016.5.5.465-469

Cited by 2 publications

(3 citation statements)

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“…We were therefore motivated to express and test the much smaller Bacillus megaterium tyrosinase (bmTYR). 65,66 This 35.5 kDa protein is robustly expressed in BL21 (DE3) E. coli with a yield of up to 160 mg/ L and has a much more accessible active site in comparison to abTYR. Gratifyingly, all tyrosine-tagged Protein L variants exposed to bmTYR were quantitatively oxidized and reacted with 150 μM aniline with over 90% conversion in 1 h, while the nontagged variant remained untouched (Figure 5b and Supporting Information, Figures S12 and S13).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…While in principle it should be possible to generate an abTYR-oxidizable variant by continuing to extend the C-terminal linker, longer linkers increase the risk of the tag interfering with protein function, and are more difficult to install during gene construction, dampening the convenience of the tyrosine tagging approach. We were therefore motivated to express and test the much smaller Bacillus megaterium tyrosinase (bmTYR). , This 35.5 kDa protein is robustly expressed in BL21 (DE3) E. coli with a yield of up to 160 mg/L and has a much more accessible active site in comparison to abTYR. Gratifyingly, all tyrosine-tagged Protein L variants exposed to bmTYR were quantitatively oxidized and reacted with 150 μM aniline with over 90% conversion in 1 h, while the nontagged variant remained untouched (Figure b and Supporting Information, Figures S12 and S13).…”

Section: Results and Discussionmentioning

confidence: 99%

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Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

et al. 2020

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Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidation of phenol-tagged cargo molecules is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepared and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chemistry for the activation of tyrosine residues that project into solution from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner and expands the scope of the reaction to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The efficacy of several secondary amines and aniline derivatives was evaluated in the coupling reactions, providing important information for coupling partner design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

et al. 2020

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“…Initially, dummy atoms were added around the copper atoms, using the UCSF Chimera [ 40 ]. The protonation states of the ionizable residues were analyzed in the PROPKA server [ 41 ], using pH 6.8, which is within the optimum range for the enzyme [ 42 ]. In the preparation stage, the OPLSAA force field was applied to treat the systems [ 43 ], then it was solvated in a 20 Å spherical water-box using the TIP3P model and the temperature (300 K) and pressure (1 bar) control were maintained with a Berendsen thermostat and pressure algorithm of Q program, respectively [ 44 ].…”

Section: Methodsmentioning

confidence: 99%

Analysis of Kojic Acid Derivatives as Competitive Inhibitors of Tyrosinase: A Molecular Modeling Approach

et al. 2021

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Tyrosinases belong to the functional copper-containing proteins family, and their structure contains two copper atoms, in the active site, which are coordinated by three histidine residues. The biosynthesis of melanin in melanocytes has two stages depending on the actions of the natural substrates L-DOPA and L-tyrosine. The dysregulation of tyrosinase is involved in skin cancer initiation. In the present study, using molecular modeling tools, we analyzed the inhibition activity of tyrosinase activity using kojic acid (KA) derivatives designed from aromatic aldehydes and malononitrile. All derivatives showed conformational affinity to the enzyme active site, and a favorable distance to chelate the copper ion, which is essential for enzyme function. Molecular dynamics simulations revealed that the derivatives formed promising complexes, presenting stable conformations with deviations between 0.2 and 0.35 Å. In addition, the investigated KA derivatives showed favorable binding free energies. The most stable KA derivatives showed the following binding free energies: −17.65 kcal mol−1 (D6), −18.07 kcal mol−1 (D2), −18.13 (D5) kcal mol−1, and −10.31 kcal mol−1 (D4). Our results suggest that these derivatives could be potent competitive inhibitors of the natural substrates of L-DOPA (−12.84 kcal mol−1) and L-tyrosine (−9.04 kcal mol−1) in melanogenesis.

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Characterization of Tyrosinase Enzyme From Native Bacillus Megaterium Sp. Strain M36

Abstract: Keywords: Melanin, monophenolase, diphenolase, TLC

Cited by 2 publications

References 2 publications

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

Analysis of Kojic Acid Derivatives as Competitive Inhibitors of Tyrosinase: A Molecular Modeling Approach

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