Structural studies of type 3 copper proteins

Polyphenol oxidases are ubiquitous among a wide range of organisms from bacteria to fungi, plant and also mammals. The function of these proteins is oxidation of phenolic substrates (tyrosinase, catechol oxidase and aureusidin synthase). These enzymes contain a type III copper center in their active site. Polyphenol oxidases are expressed in vivo in their latent state and further processed to their active state. Tyrosinase catalyzes two inducing reactions for the formation of the brown colored pigments (e.g. melanin). Aureusidin synthase catalyzes the hydroxylation and oxidation of o-diphenolic chalcones to the corresponding aurones. These aurones are responsible for the yellow flower coloration of asteraceae. The project is designed to make a contribution towards the fundamental molecular understanding on the function of polyphenol oxidases. The aims of this study is to elucidate the crystal structure of several differing polyphenol oxidase isoforms, namely tyrosinase (Agaricus bisporus) and aureusidin synthase (Coreopsis lanceolata) in their latent, intermediate and their proteolytic activated form. For the analysis of the structure and the activation of polyphenoloxidases a combinaton of protein purification, biochemical characterization, molecular biology and X- ray structure determination are the methods of choice. Recombinant expressed tyrosinase and aureusidin synthase will allow higher concentration of pure enzyme. Currently five sequences (PPO 1 - 5) encoding a tyrosinase originating of Agaricus bisporus genome are known. Only the active form (consisting residue 2-392 out of 576) of PPO3 is crystallized so far. Therefore PPO 1, 2, 4 and 5 in their active state and PPO 1-5 in their latent state, respectively, are still lacking their sufficient characterization. Sequence analyses clearly show that the taxonomical relations, length, glycosylation sites etc. differ significantly for the mushroom tyrosinase. In particular PPO4 is of special interest, because it has a 35 amino acid longer C-terminal part. This part of the enzyme shows a very hydrophobic amino acid composition and is our main target of interest. The structure of aureusisin synthase is not available up to date. Aureusidin synthase crystals soaked with different substrate homologues will address why aureusidin synthase shows an extraordinary high substrate specificity in contrast to other structural known catechol oxidases and how enzymes use structural aspects to introduce high selectivity. We expect evidences for the correctness or incorrectness of the different proposed and particularised reaction mechanisms of polyphenol oxidases.

Polyphenol oxidases (PPOs) represent a family of type 3-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 catalyze the synthesis of aurones from chalcones and are involved in yellow coloration of many ornamental plants. Our research goals were the high-yield production and biochemical and structural characterization of mushroom PPO from Agaricus bisporus (abPPO4) and AUS from Coreopsis grandiflora (cgAUS1) in order to gain knowledge about the maturation process and catalytic mechanism of PPOs as these enzymes are in vivo expressed as latent proenzymes. We developed efficient extraction and purification protocols for the isolation of both enzymes in a pure and contaminant-free form the respective natural source. Later, we also established protocols for the high-yield expression and purification of both enzymes in a recombinant form yielding sufficient amounts for further characterization. Both enzymes were biochemically characterized in their latent and active form revealing their exact cleavage site, where the enzymes active side shielding domain is cut off, and substrate specificity. Kinetic characterization of cgAUS1, revealed that AUS differs in various aspects from this enzyme class and therefore cgAUS was finally defined as aurone synthase making it a special subgroup of PPOs. Interestingly, cgAUS, which was believed to lack hydroxylase activity like COs, as it is unable to convert typical tyrosinase substrates (e.g. tyrosine), was active on its natural substrate isoliquiritigenin, which is a monophenol. Crystallization experiments led to the first crystal structure of a latent plant PPO (cgAUS) and to the first reported heterogenous crystal containing both the latent and active form of a mushroom PPO (abPPO4) within one single crystal structure. Crystal structure analysis revealed that all structurally known PPOs possess a very similar active sites making it difficult to derive any structural feature that determines the enzymes substrate specificity. However, molecular dynamics studies on cgAUS1 with different substrates revealed that amino acids located at the active site entrance are decisive for substrate specificity as they depending on the structure of the substrate stabilize or destabilize its position within the active site. Therefore, we proposed a novel mechanism for the catalytic reaction of PPOs focusing on these substrate stabilizing/destabilizing residues.