Die Erzeugung von Tyrosinaseaktivität in einer Catecholoxidase erlaubt die Identifizierung der für die C‐H‐Aktivierung in Typ‐III‐Kupferenzymen verantwortlichen Aminosäurereste

Kampatsikas, Ioannis; Pretzler, Matthias; Rompel, Annette

doi:10.1002/ange.202008859

Cited by 2 publications

(1 citation statement)

References 42 publications

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“…Heterologously expressed Sl PPO1 and Sl PPO2 from Solanum lycopersicum 17 were reported to generate the fully saturated oxy form at 25 and 11 equivalents of H 2 O 2 addition, respectively. Moreover, the addition of H 2 O 2 induced the wildtype Cg AUS from Coreopsis grandiflora that was heterologously expressed in E. coli to fully form an oxy -adduct at 24 equivalents of H 2 O 2 92 , 93 . The present study is the first to investigate the oxy -form formation of a heterologously expressed prepro-PPO which still has its signal peptides attached.…”

Section: Resultsmentioning

confidence: 99%

Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency

Ruckthong

Pretzler

Kampatsikas

et al. 2022

Sci Rep

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The “dragon-eye” fruits produced by the tropical longan tree are rich in nutrients and antioxidants. They suffer from post-harvest enzymatic browning, a process for which mainly the polyphenol oxidase (PPO) family of enzymes is responsible. In this study, two cDNAs encoding the PPO have been cloned from leaves of Dimocarpus longan (Dl), heterologously expressed in Escherichia coli and purified by affinity chromatography. The prepro-DlPPO1 contains two signal peptides at its N-terminal end that facilitate transportation of the protein into the chloroplast stroma and to the thylakoid lumen. Removal of the two signal peptides from prepro-DlPPO1 yields pro-DlPPO1. The prepro-DlPPO1 exhibited higher thermal tolerance than pro-DlPPO1 (unfolding at 65 °C vs. 40 °C), suggesting that the signal peptide may stabilize the fold of DlPPO1. DlPPO1 can be classified as a tyrosinase because it accepts both monophenolic and diphenolic substrates. The pro-DlPPO1 exhibited the highest specificity towards the natural diphenol (–)-epicatechin (kcat/KM of 800 ± 120 s−1 mM−1), which is higher than for 4-methylcatechol (590 ± 99 s−1 mM−1), pyrogallol (70 ± 9.7 s−1 mM−1) and caffeic acid (4.3 ± 0.72 s−1 mM−1). The kinetic efficiencies of prepro-DlPPO1 are 23, 36, 1.7 and 4.7-fold lower, respectively, than those observed with pro-DlPPO1 for the four aforementioned diphenolic substrates. Additionally, docking studies showed that (–)-epicatechin has a lower binding energy than any other investigated substrate. Both kinetic and in-silico studies strongly suggest that (–)-epicatechin is a good substrate of DlPPO1 and ascertain the affinity of PPOs towards specific flavonoid compounds.

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Section: Resultsmentioning

confidence: 99%

Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency

Ruckthong

Pretzler

Kampatsikas

et al. 2022

Sci Rep

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Similar but Still Different: Which Amino Acid Residues Are Responsible for Varying Activities in Type‐III Copper Enzymes?

Kampatsikas

Rompel

2020

ChemBioChem

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Type‐III copper enzymes like polyphenol oxidases (PPOs) are ubiquitous among organisms and play a significant role in the formation of pigments. PPOs comprise different enzyme groups, including tyrosinases (TYRs) and catechol oxidases (COs). TYRs catalyze the o‐hydroxylation of monophenols and the oxidation of o‐diphenols to the corresponding o‐quinones (EC 1.14.18.1). In contrast, COs only catalyze the oxidation of o‐diphenols to the corresponding o‐quinones (EC 1.10.3.1). To date (August 2020), 102 PDB entries encompassing 18 different proteins from 16 organisms and several mutants have been reported, identifying key residues for tyrosinase activity. The structural similarity between TYRs and COs, especially within and around the active center, complicates the elucidation of their modes of action on a structural basis. However, mutagenesis studies illuminate residues that influence the two activities and show that crystallography on its own cannot elucidate the enzymatic activity mode. Several amino acid residues around the dicopper active center have been proposed to play an essential role in the two different activities. Herein, we critically review the role of all residues identified so far that putatively affect the two activities of PPOs.

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Die Erzeugung von Tyrosinaseaktivität in einer Catecholoxidase erlaubt die Identifizierung der für die C‐H‐Aktivierung in Typ‐III‐Kupferenzymen verantwortlichen Aminosäurereste

Cited by 2 publications

References 42 publications

Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency

Biochemical characterization of Dimocarpus longan polyphenol oxidase provides insights into its catalytic efficiency

Similar but Still Different: Which Amino Acid Residues Are Responsible for Varying Activities in Type‐III Copper Enzymes?

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