Plant Molecular Biology

, Volume 96, Issue 1–2, pp 151–164 | Cite as

A specific amino acid residue in the catalytic site of dandelion polyphenol oxidases acts as ‘selector’ for substrate specificity

  • Sarah M. Prexler
  • Ratna Singh
  • Bruno M. Moerschbacher
  • Mareike E. Dirks-Hofmeister


Key message

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 HB2+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-I244R and PPO-6-R254I, 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.


Plant polyphenol oxidases Substrate specificity Enzyme engineering Structure–function relationship Kinetic characterization In silico docking 



Sarah M. Prexler greatly appreciates sponsoring by the Scholarship Program of the German Federal Environmental Foundation (Deutsche Bundesstiftung Umwelt, Osnabrück, Germany). The authors thank Celeste R. Brennecka, PhD from the Science Writing Support Service of the University of Münster for her editorial support.

Author contributions

SMP, MEDH, RS and BMM designed the experiments. SMP performed the bench experiments as well as the statistics, wrote the manuscript and prepared figures and tables (except Figs 1b, 4 and 6). RS performed the in silico docking studies and prepared Figs 1b, 4 and 6. All authors discussed the results, reviewed the manuscript and gave final approval for publication.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

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Supplementary material 1 (PSE 790 KB)
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Supplementary material 2 (PSE 791 KB)
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Supplementary material 3 (PSE 893 KB)
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Supplementary material 4 (PSE 863 KB)
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Supplementary material 5 (XLSX 794 KB)
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Supplementary material 6 (DOCX 14234 KB)


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.Institute for Biology and Biotechnology of PlantsUniversity of MünsterMünsterGermany
  2. 2.WeissBioTech GmbHAschebergGermany

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