Catechol oxidases (COs) and tyrosinases (TYRs) are both polyphenol oxidases (PPOs) that catalyze the oxidation of ortho‐diphenols to the corresponding quinones. By the official classification, only TYRs can also catalyze the hydroxylation of monophenols to ortho‐diphenols. Researchers have been trying to find the molecular reason for the mono‐/diphenolase specificity for decades. However, the hypotheses for the lack of monophenolase activity of plant COs are only based on crystal structures so far. To test these hypotheses, we performed site‐directed mutagenesis studies and phylogenetic analyses with dandelion PPOs offering high phylogenetic diversity, the results of which refute the structure‐based hypotheses. While plant PPOs of phylogenetic group 2 solely exhibit diphenolase activity, plant PPOs of phylogenetic group 1 unexpectedly 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.
Successful site-directed mutagenesis combined with in silico modeling and docking studies for the first time offers experimental proof of the role of the 'substrate selector' residue in plant polyphenol oxidases. The plant and fungi enzymes responsible for tissue browning are called polyphenol oxidases (PPOs). In plants, PPOs often occur as families of isoenzymes which are differentially expressed, but little is known about their physiological roles or natural substrates. In a recent study that explored these structure-function relationships, the eleven known dandelion (Taraxacum officinale) PPOs were shown to separate into two different phylogenetic groups differing in catalytic cavity architecture, kinetic parameters, and substrate range. The same study proposed that the PPOs' substrate specificity is controlled by one specific amino acid residue positioned at the entrance to the catalytic site: whereas group 1 dandelion PPOs possess a hydrophobic isoleucine (I) at position H+1, group 2 PPOs exhibit a larger, positively charged arginine (R). However, this suggestion was only based on bioinformatic analyses, not experiments. To experimentally investigate this hypothesis, we converted group 1 ToPPO-2 and group 2 ToPPO-6 into PPO-2-IR and PPO-6-RI, respectively, and expressed them in E. coli. By performing detailed kinetic characterization and in silico docking studies, we found that replacing this single amino acid significantly changed the PPO's substrate specificity. Our findings therefore proof the role of the 'substrate selector' in plant PPOs.
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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