Polyphenol oxidases (PPOs) are ubiquitously distributed among plants, bacteria, fungi and animals. They catalyze the hydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols (diphenolase activity) to o-quinones. PPOs are commonly present as an isoenzyme family. In walnut (Juglans regia), two different genes (jrPPO1 and jrPPO2) encoding PPOs have been identified. In this study, jrPPO2 was, for the first time, heterologously expressed in E. coli and characterized as a tyrosinase (TYR) by substrate scope assays and kinetic investigations, as it accepted tyramine and L-tyrosine as substrates. Moreover, the substrate acceptance and kinetic parameters (k(cat) and K-m values) towards 16 substrates naturally present in walnut were assessed for jrPPO2 (TYR) and its isoenzyme jrPPO1 (TYR). The two isoenzymes prefer different substrates, as jrPPO1 shows a higher activity towards monophenols, whereas jrPPO2 is more active towards o-diphenols. Molecular docking studies performed herein revealed that the amino acid residue in the position of the 1st activity controller (His(B1)+1; in jrPPO1 Asn240 and jrPPO2 Gly240) is responsible for the different enzymatic activities. Additionally, interchanging the 1st activity controller residue of the two enzymes in two mutants (jrPPO1-Asn240Gly and jrPPO2-Gly240Asn) proved that the amino acid residue located in this position allows plants to selectively target or dismiss substrates naturally present in walnut.