Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

Abstract

Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1.