Identification of the amino acid position controlling the different enzymatic activities in walnut tyrosinase isoenzymes ( jr PPO1 and jr PPO2)

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 ( jr PPO1 and jr PPO2) encoding PPOs have been identified. In this study, jr PPO2 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 jr PPO2 (TYR) and its isoenzyme jr PPO1 (TYR). The two isoenzymes prefer different substrates, as jr PPO1 shows a higher activity towards monophenols, whereas jr PPO2 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 jr PPO1 Asn240 and jr PPO2 Gly240) is responsible for the different enzymatic activities. Additionally, interchanging the 1st activity controller residue of the two enzymes in two mutants ( jr PPO1-Asn240Gly and jr PPO2-Gly240Asn) proved that the amino acid residue located in this position allows plants to selectively target or dismiss substrates naturally present in walnut.