Structural Diversity in the Dandelion (Taraxacum officinale) Polyphenol Oxidase Family Results in Different Responses to Model Substrates

Dirks‐Hofmeister, Mareike E.; Singh, Ratna; Leufken, Christine M.; Inlow, Jennifer K.; Moerschbacher, Bruno M.

doi:10.1371/journal.pone.0099759

Cited by 30 publications

(27 citation statements)

References 58 publications

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“…To biochemically assess the molecular basis for mono‐/diphenolase specificity, we performed site‐directed mutagenesis and detailed kinetic studies with well‐characterized dandelion ( Taraxacum officinale ) PPOs. Dandelion PPOs form one of the largest PPO families ever discovered consisting of eleven isoenzymes separating into two different phylogenetic groups (group 1 and group 2) . Recently, it has been proposed that plant PPOs in general divide into these two groups whose members differ in catalytic cavity architecture, kinetic parameters, and substrate specificity.…”

Section: Methodsmentioning

confidence: 99%

Catechol Oxidase versus Tyrosinase Classification Revisited by Site‐Directed Mutagenesis Studies

et al. 2019

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Catechol oxidases (COs) and tyrosinases (TYRs) are both polyphenol oxidases (PPOs) that catalyze the oxidation of ortho-diphenols to the corresponding quinones.B yt he official classification, only TYRs can also catalyze the hydroxylation of monophenols to ortho-diphenols.R esearchers have been trying to find the molecular reason for the mono-/diphenolase specificity for decades.H owever,t he hypotheses for the lacko fm onophenolase activity of plant COs are only based on crystal structures so far.T ot est these hypotheses,w ep erformed site-directed mutagenesis studies and phylogenetic analyses with dandelion PPOs offering high phylogenetic diversity,the results of which refute the structurebased hypotheses.W hile plant PPOs of phylogenetic group 2 solely exhibit diphenolase activity,plant PPOs of phylogenetic group 1u nexpectedly also show monophenolase activity.This finding sheds new light upon the molecular basis for mono-/ diphenol substrate specificity and challenges the current practice of generally naming plant PPOs as COs.Catechol oxidases (COs;E C1 .10.3.1) and tyrosinases (TYRs;E C1 .14.18.1) are ubiquitously distributed enzymes that are,for example,responsible for the undesired browning of damaged fruits and vegetables.A lthough they are of significant interest for biotechnological applications,t he molecular basis for their different activities-either only on ortho-diphenols (COs) or additionally on monophenols (TYRs;Scheme 1)-is still unknown. Scheme 1. Reactionsc atalyzed by catechol oxidases and tyrosinases.[*] Dr.

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Section: Methodsmentioning

confidence: 99%

Catechol Oxidase versus Tyrosinase Classification Revisited by Site‐Directed Mutagenesis Studies

et al. 2019

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“…S1). However, the sequence of ToPPO-5 (TYR) 60 and mutants of dandelion PPO (ToPPO-2) 32 prove that TYRs can still function on monophenols, even though Thr is present at the 1 st activity controller position. Kinetic data of the herein presented jrPPO1 mutant Leu244Arg demonstrate that Arg at the 2 nd activity controller position does not convert a TYR into a CO either.…”

Section: Kinetic Characterization Of Recombinant Jrppo1-wt Recombinamentioning

confidence: 99%

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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“…[20] Based on these studies we were able to produce l-VvPPOcs-3 heterologously after some modification. Cabernet Sauvignon), followed by first strand cDNA( complementary DNA) synthesis,cloning, and sequencing.Lately, the Dirks-Hofmeister/Moerschbacher group established an expression system for plant PPOs in E. coli.…”

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confidence: 98%

Tyrosinase versus Catechol Oxidase: One Asparagine Makes the Difference

2016

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Tyrosinases mediate the ortho-hydroxylation and two-electron oxidation of monophenols to ortho-quinones. Catechol oxidases only catalyze the oxidation of diphenols. Although it is of significant interest, the origin of the functional discrimination between tyrosinases and catechol oxidases has been unclear. Recently, it has been postulated that a glutamate and an asparagine bind and activate a conserved water molecule towards deprotonation of monophenols. Here we demonstrate for the first time that a polyphenoloxidase, which exhibits only diphenolase activity, can be transformed to a tyrosinase by mutation to introduce an asparagine. The asparagine and a conserved glutamate are necessary to properly orient the conserved water in order to abstract a proton from the monophenol. These results provide direct evidence for the crucial importance of a proton shuttle for tyrosinase activity of type 3 copper proteins, allowing a consistent understanding of their different chemical reactivities.

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Structural Diversity in the Dandelion (Taraxacum officinale) Polyphenol Oxidase Family Results in Different Responses to Model Substrates

Cited by 30 publications

References 58 publications

Catechol Oxidase versus Tyrosinase Classification Revisited by Site‐Directed Mutagenesis Studies

Catechol Oxidase versus Tyrosinase Classification Revisited by Site‐Directed Mutagenesis Studies

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

Tyrosinase versus Catechol Oxidase: One Asparagine Makes the Difference

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