Eine peptidvermittelte Selbstspaltungsreaktion initiiert die Tyrosinaseaktivierung

Kampatsikas, Ioannis; Bijelic, Aleksandar; Pretzler, Matthias; Rompel, Annette

doi:10.1002/ange.201901332

Cited by 5 publications

(4 citation statements)

References 31 publications

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“…Using the monophenolic substrate tyramine, activity was once again observed only after addition of SDS. Therefore, the interactions in-between the two domains appear to be of significant strength and probably resemble the interactions in-between the active and C-terminal domain of MdPPO1 where a self-cleavage reaction in-between those domains did also result in a cleaved but still latent enzyme 16 . Activation of jrPPO1-wt was tested using different molarities of salts (NaCl, KCl, MgCl 2 and CaCl 2 ), all of which resulted in active enzyme with the divalent ions (Mg 2+ and Ca 2+ ) requiring lower molarities than the monovalent ions (Na + and K + ) ( Fig.…”

Section: Resultsmentioning

confidence: 98%

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Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

Panis

Kampatsikas

Bijelic

et al. 2020

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polyphenol oxidases (ppos) comprise tyrosinases (tYRs) and catechol oxidases (cos), which catalyse the initial reactions in the biosynthesis of melanin. tYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas cos react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/ Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono-versus diphenolase activity in the type-iii copper enzyme jrPPO1.Tyrosinases (TYRs), catechol oxidases (COs) and aurone synthases (AUSs) represent the polyphenol oxidase (PPO) family, which is an umbrella term for copper metalloenzymes 1-3 containing one type-III copper center. TYRs catalyse the hydroxylation of monophenols to o-diphenols (EC 1.14.18.1, monophenolase activity) as well as the subsequent oxidation of o-diphenols to their corresponding o-quinones (EC 1.10.3.1, diphenolase activity) 1,4 , whereas COs catalyse only the latter reaction, unable to react with monophenolic substrates (Fig. 1). AUSs participate in the formation of aurones from chalcone precursors and are involved in plant secondary metabolism 5,6 . Quinones produced by PPOs usually undergo non-enzymatic reactions, polymerize 7 and finally form melanin products 8,9 . PPOs occur in a broad spectrum of organisms, including archaea 10 , bacteria 11 , fungi 2 , plants 8 and animals 12,13 . In plants, they are believed to be involved in defence mechanisms associated with the formation of browning substances, which is triggered by mechanical damage or wounding 14 , while in animals their reaction products are responsible for coloring of skin, hair and eyes 13 .TYR from Juglans regia (walnut, jrPPO1) is expressed in vivo as a latent 66.8 kDa pro-enzyme consisting of three domains 15 : an N-terminal chloroplast transit peptide (~12 kDa) 15 , the catalytically active domain (~39 kDa) and the C-terminal domain (~16 kDa) that shields the entrance to the catalytic pocket and keeps the enzyme in a latent state. In vivo enzymatic activity is triggered by the removal of the C-terminal domain 16 . Alternati...

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Section: Resultsmentioning

confidence: 98%

“…Removing the C-terminal domain by extensive proteolytic cleavage seems to be a prerequisite to convert the enzyme from its latent state into the active form. Otherwise, the still intact C-terminal domain is presumably held in place by non-covalent forces, blocking access to the active center (pre-active form) 16 .…”

Section: Resultsmentioning

confidence: 99%

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

Panis

Kampatsikas

Bijelic

et al. 2020

Sci Rep

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“…At the beginning, the pipeline searched for an appropriate template of the investigated sequence (OU702517) based on BLAST 105 and HHblits 106 . The search resulted in Md PPO1 66 , Malus domestica TYR 1 (PDB: 6ELS) 35 , 36 , as the hit which exhibited the highest coverage value (0.86, with 69.80% sequence identity). The final homology model of the pro- Dl PPO1 was then created using the 6ELS structure as the template and visualization was done using the PyMol Molecular graphic system (Schrödinger, LLC) 107 .…”

Section: Methodsmentioning

confidence: 99%

“…sodium dodecyl sulfate, SDS) 17 , 27 – 33 . Although the natural proteolytic mechanism is unknown 17 , it has recently been reported that plant PPOs are capable of self-activation which can autoproteolytically separate the active enzyme from the C-terminal domain 34 – 36 .…”

Section: Introductionmentioning

confidence: 99%

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

Ruckthong

Pretzler

Kampatsikas

et al. 2022

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

2020

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Tyrosinasen (TYRs) katalysieren die Hydroxylierung von Phenolen und die Oxidation der resultierenden o‐Diphenole zu o‐Chinonen, während Catecholoxidasen (COs) nur die letztere Aktivität aufweisen. Die Auronsynthase (AUS) reagiert nicht mit klassischen Tyrosinasesubstraten wie Tyramin und l‐Tyrosin, jedoch hydroxyliert sie ihr natürliches Substrat: das Isoliquiritigenin. Der strukturelle Unterschied zwischen TYRs, COs und AUS, der zu ihren unterschiedlichen katalytischen Aktivitäten führt, ist noch immer ein Rätsel. Zu dessen Klärung wurde eine Bibliothek von 39 Mutanten der AUS von Coreopsis grandiflora (CgAUS) erstellt. Aktivitätsstudien an diesen zeigten, dass die Reaktivität der drei konservierten Histidine (HisA2, HisB1 und HisB2) durch ihre benachbarten Reste (HisB1+1, HisB2+1) und den Wasserträgerrest bestimmt wird, womit diese entweder als stärkere Basen reagieren oder/und in einer für das Verschieben von Substratprotonen günstigen Position stabilisiert werden. Diese Studie liefert das Verständnis für die CH‐Aktivierung basierend auf dem Typ‐III‐Kupferzentrum, das in zukünftigen biotechnologischen Prozessen verwendet werden kann.

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Eine peptidvermittelte Selbstspaltungsreaktion initiiert die Tyrosinaseaktivierung

Cited by 5 publications

References 31 publications

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

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

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

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