Computational and structural characterization of engineered Type-3-copper variants

Abstract

This master thesis aimed to use experiments at the laboratory and computational chemistry to begin the process of characterizing suitable enzyme candidates for conversion of CO2 to oxalate by type- 3 copper proteins. The WT of Tyrosinase (TyrBm) from Bacillus megaterium and mutants of this protein F227Y and F65Y were characterized through expression, purification, activity assays, and X-ray crystallography. The diffraction data from the X-ray experiments were processed, and the resulting structure was further analyzed by computational chemistry using the software xTB -GFN -FF. The analysis was performed to see if xTB -GFN -FF was able to reproduce and predict variants of the enzyme structure. The experiments showed that both TyrBm WT and the mutants were successfully expressed and purified for further activity assays and crystallization. Activity assays were performed using caffeic acid as the substrate and showed that copper ions had to be added to the enzymes prior to the assay for the mutants to show activity. The loss of activity indicated that copper ions had been lost in the active site during the purification process. Crystals of TyrBm WT and the mutants F65Y and F227Y were obtained using the hanging drop method, and the crystal structure of TyrBm F227Y crystallized in the presence of zinc ions were solved using X-ray crystallography. The enzyme crystallized as a dimer in the asymmetric unit, with six histidine residues coordinated around two metal ions. As the enzyme was crystallized in the presence of zinc ions, there was uncertainty if copper or zinc was present in the active site. The enzyme was therefore geometry optimized with xTB -GFN -FF with Cu(I), Cu(II), and Zn(II), respectively, in the active site with H2O, then OH- and finally O2- bridging the metal ions. The result showed that xTB -GFN -FF could reproduce and model the different enzyme variants at a reasonable accuracy. The model with Zn(II) and H2O seemed to be most similar to the structure from the X-ray crystallization and therefore substantiate the hypothesis that zinc and not copper were in the model´s active site. The work done in this thesis will provide some of the groundwork to enable further COOFIX research for “Evolving novel carbon dioxide reducing enzymes”.

Målet med denne masteroppgaven var å bruke eksperimentelle metoder på laboratoriet og beregningsorientert kjemi for å begynne karakteriseringen av mulige enzym kandidater for konvertering av CO2 til oxalate ved hjel av kobber -3 proteiner. Villtype av Tyrosinase fra Bacillus Megaterium (TyrBm WT) og mutanter av dette proteinet F227Y og F65Y ble karakterisert gjennom uttrykking, rensing, aktivitet assay og røntgenkrystallografi. Diffraksjonsdata fra røntgenkrystallografieksperimentene ble deretter prosessert, og den endelige strukturen ble videre analysert ved å bruke beregningsorientert kjemi med programvaren, xTB -GFN -FF. Analysen ble gjort for å finne ut om xTB -GFN -FF er kapabel til å reprodusere og predikere varianter av enzym strukturen.