Tyrosinases and catechol oxidases belong to the polyphenol oxidase (PPO) enzyme family, which is mainly responsible for the browning of fruits. Three cDNAs encoding PPO pro-enzymes have been cloned from leaves of Malus domestica (apple, MdPPO). The three pro-enzymes MdPPO1-3 were heterologously expressed in E. coli yielding substantial amounts of protein and have been characterized with regard to their optimum of activity resulting from SDS, acidic and proteolytic activation. Significant differences were found in the kinetic characterization of MdPPO1-3 when applying different mono- and diphenolic substrates. All three enzymes have been classified as tyrosinases, where MdPPO1 exhibits the highest activity with tyramine (kcat = 9.5 s−1) while MdPPO2 and MdPPO3 are also clearly active on this monophenolic substrate (kcat = 0.92 s−1 and kcat = 1.0 s−1, respectively). Based on the activity, sequence data and homology modelling it is proposed that the monophenolase and diphenolase activity of PPOs can be manipulated by the appropriate combination of two amino acids, which are located within the active site cleft and were therefore named “activity controllers”.

HighlightsThe first PPO gene (cgAUS1) involved in 4-deoxyaurone formation is identified.AUS1 is expressed as latent pro-enzyme in E. coli and purified to homogeneity.Diphenolase activity of AUS1 pro-enzyme is proven using SDS as an activation agent.Gene expression studies suggest a physiological role for AUS1 in aurone formation.

Polyphenol oxidases (PPOs) contain the structurally similar enzymes tyrosinases (TYRs) and catechol oxidases (COs). Two cDNAs encoding pro-PPOs from tomato (Solanum lycopersicum) were cloned and heterologously expressed in Escherichia coli. The two pro-PPOs (SlPPO1-2) differ remarkably in their activity as SlPPO1 reacts with the monophenols tyramine (kcat = 7.94 s−1) and phloretin (kcat = 2.42 s−1) and was thus characterized as TYR, whereas SlPPO2 accepts only diphenolic substrates like dopamine (kcat = 1.99 s−1) and caffeic acid (kcat = 20.33 s−1) rendering this enzyme a CO. This study, for the first time, characterizes a plant TYR and CO originating from the same organism. Moreover, X-ray structure analysis of the latent holo- and apo-SlPPO1 (PDB: 6HQI and 6HQJ) reveals an unprecedented high flexibility of the gatekeeper residue phenylalanine (Phe270). Docking studies showed that depending on its orientation the gatekeeper residue could either stabilize and correctly position incoming substrates or hinder their entrance into the active site. Furthermore, phloretin, a substrate of SIPPO1 (Km = 0.11 mM), is able to approach the active centre of SlPPO1 with both phenolic rings. Kinetic and structural results indicate that phloretin could act as a natural substrate and connote the participation of PPOs in flavonoid-biosynthesis.

Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO4 and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (KM = 2.0 mM) and chlorogenic acid (KM = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103) by means of peptide mass fingerprinting.