Developing de novo designed models of the type III copper proteins

Catechol Oxidase and tyrosinase are prominent members of the type III copper (T3Cu) proteins and they play a key role in diverse biological processes [1]. The T3Cu site, two magnetically coupled cop- per ions, allows the binding and subsequent activation of molecular oxygen. Tyrosinase catalyzes the orthohydroxylation of monophenol (monophenolase activity) and the subsequent oxidation of the diphenolic product to the quinone (diphenolase activity), whereas catechol oxidase shows dipheno- lase, but lacks monooxygenase activity. X-ray crystallographic investigations of the native enzymes suggest that the monophenolase activity is possible only in presence of a structural water, whose role is the deprotonation of the phenolic substrate. However, the structural determinants to differentiate the two classes of enzymes are still unclear [2]. De novo protein design is one of the approaches of choice to study metalloenzymes. A polypeptide sequence that is not directly related to any natural protein allows the elucidation of unbiased structural determinants. Indeed, employing this approach, we have already developed the DF proteins, a series of artificial metalloenzymes, that recapitulate not only the structural, but also the functional features of the natural diiron-oxo-proteins [3]. Here, we describe the development of DR1, a de novo designed metalloprotein inspired by the T3Cu proteins. The computational design consisted primarily in the positioning of the first coordination sphere to recreate the T3Cu site. Subsequently, the second coordination sphere and the hydrophobic core were optimized to stabilize the metal-binding site and the intended native-like tertiary structure. Finally, the overall stability of the final model was evaluated using molecular dynamics simulations. Our preliminary characterization demonstrates that DR1 binds two copper ions as designed and, more importantly, catalyzes the oxidation of 3,5-di-tert-butyl catechol to the o-quinone, showing diphenolase activity. Starting from DR1, new models will be designed to unveil the structural determinants to induce the monophenolase activity. References: [1] Kaintz, C.; Mauracher, S. G.; Rompel, A. Chapter One - Type-3 Copper Proteins: Recent Advances on Polyphenol Oxidases. Christov, C. Z.; Advances in Protein Chemistry and Structural Biology; Academic Press: 2014; Vol. 97. [2] Decker, H.; Solem, E.; Tuczek, F. Inorganica Chimica Acta 2018, 481, 32. [3] Chino, M.; Maglio, O.; Nastri, F.; Pavone, V.; DeGrado, W. F.; Lombardi, A. European Journal of Inorganic Chemistry 2015, 2015 (21), 3371.