Type 3 copper proteins

Polyphenol oxidases (PPOs) represent a family of type-III-copper metalloenzymes, which include tyrosinases (TYRs), catechol oxidases (COs) and aurone synthases (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 catalyze the synthesis of aurones from chalcones and are involved in yellow coloration of many ornamental plants. Our follow-up project is focusing on PPOs from the edible mushroom Agaricus bisporus and aurone synthase from Coreopsis grandiflora. Six genes of Agaricus bisporus encode for the corresponding number of PPOs, termed AbPPO1- 6, whereas only AbPPO3 and 4 are expressed in significant amounts by the fruiting bodies of mushrooms with the latter being the most studied AbPPO in the literature. As a successful protocol for the expression of AbPPO4 already exists, the aim of the proposed AbPPO- related project is the recombinant expression of AbPPO1, 2, 3, 5 and 6 and their biochemical characterization. Furthermore, it is planned to crystallize all AbPPOs, whereby the crystal structures of AbPPO3 and 4 are already available. The biochemical characterization of these PPOs will focus on their activities in order to determine, if all AbPPOs are involved in the browning reaction of mushrooms. In this way, we hope to clarify why Agaricus bisporus requires six different PPOs. The activity studies will provide valuable information on the substrate preference of each AbPPO. Moreover, all AbPPOs will be tested for their ability to cross-link peptides which may find its later application as an underwater glue. Regarding CgAUS1, extensive mutagenesis studies will be performed mutating PPO-characteristic residues located within the second shell of the active site. Subsequent activity assays are supposed to identify amino acids that are decisive for the enzymes activity as it is expected that some mutations will convert the enzyme from a CO with hydroxylase activity to a pure CO or a pure TYR. The CgAUS1 mutant with the highest monophenolase activity will be subject to crystallization trials and the resulting crystal structure is supposed to provide structural insights into the catalytic mechanism by structural comparison between the mutant and the wild type structure. The obtained results will dramatically increase the knowledge about the catalytic mechanism of CgAUS1 and plant PPOs in general and in addition shed light on the monophenolase/diphenolase discrepancy of PPOs. Based on the biochemical and structural knowledge CgAUS1 (and its mutants), new specific inhibitors will be designed that target specific structural features which are indispensable for the enzymes catalytic activity as a new approach for a PPO inhibitor.

Polyphenol oxidases (PPOs) represent a family of type III copper metalloenzymes that 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 (diphenolase activity), while COs can only 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. Our research goals were high-yield production, biochemical and structural characterization of plant PPOs from apricot, longan and lanceolata grandiflora to gain knowledge about the structural difference in active site design of TYRs and COs. We mutated a catechol oxidase into a tyrosinase for the first time, identifying the amino acid residues responsible for tyrosinase activity und proposed a reaction mechanim for PPO activity. In addition, we have carried out inhibition of mushroom PPO applying organic and purely inorganic compounds. The complete Agaricus bisporus (AbPPO1-6) tyrosinase family was cloned from mRNA and heterologously expressed. All six isoenzymes accept a wide range of mono- and diphenolic substrates, but show marked differences in their specificity and enzymatic reaction rate. In cooperation with scientists from the HU Berlin, we have expanded the concept of artificial mussel adhesive proteins with the help of mushroom tyrosinase by integrating a sequence module for cohesion control. As environmental issues become increasingly important for the healthy survival of human kind, we investigated the role of bacterial tyrosinases in peatlands and in the geochemical cycle. To provide convincing evidence for the novel concept that bacterial tyrosinases are among the key enzymes affecting carbon cycling in wetland ecosystems, bacterial organisms native to wetland ecosystems that contain a TYR gene in their respective genomes (tyr+) were identified, yielding a phylogenetically diverse community of Tyr+ bacteria native to wetlands. After increased aeration of previously anoxic wetland soils due to climate change, TYRs are among the enzymes capable of reducing the concentration of phenolic compounds in wetland ecosystems, thereby likely destabilize large amounts of carb on stored in these ecosystems and releases it as carbon dioxide.