Matthias Pretzler

The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of "Substrate-Guiding Residues" for Enzymatic Specificity.

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. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. 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.

Heterologous expression and characterization of functional mushroom tyrosinase (AbPPO4).

Tyrosinases are an ubiquitous group of copper containing metalloenzymes that hydroxylate and oxidize phenolic molecules. In an application context the term ‘tyrosinase’ usually refers to ‘mushroom tyrosinase’ consisting of a mixture of isoenzymes and containing a number of enzymatic side-activities. We describe a protocol for the efficient heterologous production of tyrosinase 4 from Agaricus bisporus in Escherichia coli. Applying this procedure a pure preparation of a single isoform of latent tyrosinase can be achieved at a yield of 140 mg per liter of autoinducing culture medium. This recombinant protein possesses the same fold as the enzyme purified from the natural source as evidenced by single crystal X-ray diffraction. The latent enzyme can be activated by limited proteolysis with proteinase K which cleaves the polypeptide chain after K382, only one The latent enzyme can amino acid before the main in-vivo activation site. Latent tyrosinase can be used as obtained and enzymatic activity may be induced in the reaction mixture by the addition of an ionic detergent (e.g. 2 mM SDS). The proteolytically activated mushroom tyrosinase shows >50% of its maximal activity in the range of pH 5 to 10 and accepts a wide range of substrates including mono- and diphenols, flavonols and chalcones.

Fungal Tyrosinases: Why Mushrooms Turn Brown

Abstract Tyrosinases are copper-containing metalloenzymes that catalyze the reactions that provide the starting material for melanin biosynthesis. They use molecular oxygen for the ortho -hydroxylation of monophenols and the subsequent oxidation of o -diphenols to o -quinones. Tyrosinases fulfill important roles in the life cycle of fungi where they are involved in the formation of spores, do produce pigments that protect the mycelium against radiation or pathogen challenges, and may also participate in fungal pathogenesis. Their catalytic activities provide them with great potential for application in pharmaceutical, environmental, or food biotechnology. The basic characteristics of fungal tyrosinases are outlined and their major applications are presented.

Purification and Characterization of Latent Polyphenol Oxidase from Apricot (Prunus armeniaca L.)

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.

Total Synthesis, Stereochemical Assignment, and Divergent Enantioselective Enzymatic Recognition of Larreatricin

Abstract A concise and efficient total synthesis of the lignan natural product larreatricin as well as an unambiguous assignment of configuration of its enantiomers are reported, resolving a long‐held controversy. Enzyme kinetic studies revealed that different polyphenol oxidases show high and remarkably divergent enantioselective recognition of this secondary metabolite.

In crystallo activity tests with latent apple tyrosinase and two mutants reveal the importance of the mutated sites for polyphenol oxidase activity

M. domestica polyphenol oxidase 1 (MdPPO1) was recombinantly expressed in its latent form (Lys1–Ser504) and successfully mutated at four different positions around the active centre which have been proposed to be decisive for the activity of the enzyme. The wild-type MdPPO1 and two of the mutants were successfully crystallized. In crystallo activity tests demonstrated the importance of these amino acids for the activity of the enzyme.