Specialized polyphenol oxidases

Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Our group has presented the first crystal structure of a plant tyrosinase (from Juglans regia). The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed. The project is designed to make a contribution towards the fundamental molecular understanding on the function of plant originated tyrosinase and is the basis for biotechnological processes. The aim of this study is to elucidate the crystal structure of several second shell amino acid mutated forms, which are located in the second shell around the active site. For the analysis of the structure and the activation of polyphenol oxidases a combination of protein purification, biochemical characterization, molecular biology and X-ray structure determination are the methods of choice.

Polyphenol oxidases (PPOs) represent a family of type-III-copper metalloenzymes, which include tyrosinases (TYRs), catechol oxidases (COs) and aurone synthase (AUS). TYRs catalyze the ortho-hydroxylation of monophenols (monophenolase activity) and the subsequent oxidation of the resulting ortho-diphenols to ortho-quinones, whereas COs are only able to catalyze the latter reaction. The ortho-quinones are highly reactive and represent the starting material for the biosynthesis of melanin, and thus, PPOs are involved in pigment formation. AUS catalyzes the synthesis of aurones from chalcones and is engaged in the yellow coloration of many ornamental plants. Our research goals were the high -yield production and biochemical and structural characterization of plan t PPOs from walnut Juglans regia (jrPPO1), apple Malus domestica (MdPPO1-3), tomato Solanum lycopersicum (SlPPO1-2) and larreatricin hydroxylase from Larrea tridentata in order to gain knowledge about the structural difference between TYRs and COs, and the maturation process of these enzymes that are in vivo expressed as inactive proenzymes. We also established protocols for the high-yield expression and purification of walnut, tomato, and apple PPOs in a recombinant form yielding sufficient amounts for further characterization. All plant enzymes were biochemically characterized and kinetic characterization classified the enzymes as either a TYR or a CO. We identified four amino acid residues as being responsible for tyrosinase activity, namely the waterkeeper, the gatekeeper, and the two activity controllers. Besides, we worked on a novel activation process of PPOs: The structural and biochemical characterization of PPO1 from Malus domestica (MdPPO1) reveals self-cleavage as an activation route for plant PPOs. The conversion of inactive pro-polyphenol oxidases (pro- PPOs) to the active enzymes results from the proteolytic cleavage of their C-terminal domains. The sequence Lys355-Val370 located in the linker between the active and the C - terminal domain is indispensable for the self-cleaving as a mutant lacking this peptide did not undergo self-cleavage. Partial introduction (Lys352-Ala360) of this peptide into the sequences of two PPOs, MdPPO2 and aurone synthase (CgAUS1), triggered self -cleavage reaction in the resulting mutants. This is the first experimental proof of a self-cleavage inducing peptide in PPOs, unveiling a novel mode of activation for this enzyme class independent of any external protease. We developed an in crystallo activity assay using crystals of the wild type and mutants of apple tyrosinase (MdPPO1). The effects of the mutation on the enzyme activity were observed after the addition of the monophenolic substrate tyramine and the diphenolic substrate dopamine to crystal-containing drops by colour changes of the crystals, owing to the conversion of the substrates to dark chromophore products.