A specific amino acid residue in the catalytic site of dandelion polyphenol oxidases acts as ‘selector’ for substrate specificity
- 341 Downloads
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.
KeywordsPlant 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.
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.
- Chen VB, Arendall WB, Headd JJ, Keedy D, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr Sect D Biol Crystallogr 66:12–21. https://doi.org/10.1107/S0907444909042073 CrossRefGoogle Scholar
- Dirks-Hofmeister ME, Singh R, Leufken CM, Inlow JK, Moerschbacher BM (2014) Structural diversity in the dandelion (Taraxacum officinale) polyphenol oxidase family results in different responses to model substrates. PLoS ONE 9:e99759. https://doi.org/10.1371/journal.pone.0099759 CrossRefPubMedPubMedCentralGoogle Scholar
- Laskowski RA, Swindells MB (2011) LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 2778–2786. https://doi.org/10.1021/ci200227u
- Leufken CM, Moerschbacher BM, Dirks-Hofmeister ME (2015) Dandelion PPO-1/PPO-2 domain-swaps: the C-terminal domain modulates the pH optimum and the linker affects SDS-mediated activation and stability. Biochim Biophys Acta - Proteins Proteomics 1854:178–186. https://doi.org/10.1016/j.bbapap.2014.11.007 CrossRefGoogle Scholar
- Molitor C, Mauracher SG, Rompel A (2016) Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases. Proc Natl Acad Sci E1806–E1815. https://doi.org/10.1073/pnas.1523575113
- Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ, (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 19:1639–1662. 10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-B CrossRefGoogle Scholar
- Sullivan ML, Hatfield RD, Thoma SL, Samac DA (2004) Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa. Plant Physiol 136:3234–3244. https://doi.org/10.1104/pp.104.047449.nonprotein CrossRefPubMedPubMedCentralGoogle Scholar