Abstract
Polyphenol oxidases (PPOs) are ubiquitous type-3 copper enzymes that catalyze the oxygen-dependent conversion of o-diphenols to the corresponding quinones. In most plants, PPOs are present as multiple isoenzymes that probably serve distinct functions, although the precise relationship between sequence, structure and function has not been addressed in detail. We therefore compared the characteristics and activities of recombinant dandelion PPOs to gain insight into the structure–function relationships within the plant PPO family. Phylogenetic analysis resolved the 11 isoenzymes of dandelion into two evolutionary groups. More detailed in silico and in vitro analyses of four representative PPOs covering both phylogenetic groups were performed. Molecular modeling and docking predicted differences in enzyme-substrate interactions, providing a structure-based explanation for grouping. One amino acid side chain positioned at the entrance to the active site (position HB2+1) potentially acts as a “selector” for substrate binding. In vitro activity measurements with the recombinant, purified enzymes also revealed group-specific differences in kinetic parameters when the selected PPOs were presented with five model substrates. The combination of our enzyme kinetic measurements and the in silico docking studies therefore indicate that the physiological functions of individual PPOs might be defined by their specific interactions with different natural substrates.
Highlights
Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper enzymes that catalyze the oxidation of diphenols to their corresponding quinones with molecular oxygen as a co-substrate (Figure 1)
Mature PPOs consist of the active-site-containing tyrosinase domain, a short linker region, and a C-terminal domain hypothized to cover the active site of the enzyme when in its latent state [13,15,16]
Due to the twin-arginine motif (Figure 3) localization is predicted to be in the thylakoid lumen. bCalculated average molecular weight (MW) in kDa and isoelectric point of the mature protein, computed by the Compute pI/Mw tool [45,46]. cValues given in % sequence identity of full length protein sequences relative to PPO-1. dExpression was repeatedly tested by semi-quantitative reverse transcriptase (RT)-PCR; PPOs which were expressed in a given tissue in at least one experiment were assigned as ‘‘occasionally’’ expressed. doi:10.1371/journal.pone.0099759.t003
Summary
Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper enzymes that catalyze the oxidation of diphenols to their corresponding quinones with molecular oxygen as a co-substrate (Figure 1). Plant PPOs are almost always present as multiple isoenzymes, differing in catalytic activity and expression profile, potentially corresponding to their distinct biological functions [7,8,9,10,11]. Comparative sequence analyses show all plant PPOs to have in common a three-domain structure [4,12,13]. Mature PPOs consist of the active-site-containing tyrosinase domain, a short linker region, and a C-terminal domain hypothized to cover the active site of the enzyme when in its latent state [13,15,16]. The natural substrates for most plant PPOs are unknown, diphenolic flavonoids such as catechins have been proposed [25]
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