This thesis describes the characterization of the histidine brace (His-brace) from lytic polysaccharide monooxygenases (LPMOs) using biochemical, structural and functional methods. LPMOs catalyze the oxidative cleavage of glycosidic bonds. They do this by employing a copper-cofactor and a redox cycle, i.e. the copper undergoes a reduction from the Cu(II) state to the Cu(I) state. This leads to the activation of an oxygen species and hydrogen abstraction from a C-H bond. These studies were performed on a reference batch of the model AA9A obtained from the fungus Thermoascus aurantiacus (TaAA9A) which, as part of this work, was purified and carefully characterized with regard to its concentration, copper concentration and its reaction with ascorbate.
Initially, three LPMOs TaAA9A, Lentinus similis (LsAA9A) and Thielavia terrestris (TtAA9E) were combined individually with cellulases from Trichoderma reesei (Cel6A and Cel7A) to study the synergistic effects between cellulases and LPMOs in terms of their degree of synergy (DS) on amorphous and crystalline cellulose. This study showed that the three LPMOs significantly improved the hydrolytic efficiency of Cel6A, on both cellulosic substrates. The highly processive, reducing-end acting Cel7A synergized with the C1-C4 oxidizing LPMOs, TaAA9A and LsAA9A, but was inhibited by the presence of C1-oxidizing TtAA9E.
Additional His-brace proteins where expressed using a novel reinterpretation of the golden gate cloning based platform. From this production platform, one protein was purified and, together with the benchmark LPMO TaAA9A, used to assay key differences in the proteinaceous environment around the His-brace and their effect on oxidation of ascorbate. For this purpose, an absorbance based ascorbate assay was developed and used. The three different proteins studied have distinct biological roles and His-brace compositions and thus they show three different behaviors. i) TaAA9A rapidly oxidized ascorbate, but produced fewer radicals than free copper. ii) The LPMO like protein from the fungus Cryptococcus neoformans Bim1 is sensitive to H2O2, which accelerated the release of the Cu ion from the protein. iii) The Cu(II) chelator from the bacterium Pseudomonas fluorescens (PfCopC) was not redox active and did not reduce ascorbate.
To further investigate the protein ligands around the His-brace, PfCopC was mutated using a systematic approach. It was found that the coordinating residue Asp83 was indispensable for copper binding and, in contrast to previous studies, no alternative copper-binding mode could be found. Furthermore, the residue Glu27 was found to be important for chaperone function.
During the development of the assay to use phenolphthalein (PHP) as a novel glycoside mimic, it was found that TaAA9A could utilize fructose and dehydroascorbic acid (DHA) as co-substrates for cellulolytic activity. These two compounds are not normally considered to be LPMO co-substrates. This novel PHP assay improved our understanding of the LPMO reaction, and is a rare example of a finding from a high-throughput assay that translates directly into enzyme activity on an insoluble substrate. With this new assay at hand, the well-studied LPMO LsAA9A was re-investigated. It was found that LsAA9A has a strict stoichiometric requirement for H2O2 and is not catalytically competent with O2. Furthermore, a 2/3 non-oxidized to 1/3 oxidized sugar stoichiometry was found for the reaction. This finding show that the cleavage of glycosidic bonds and oxidation of the sugar are coupled in a different way than previously suggested.
Overall, the work presented in this thesis expands our current knowledge of the Cu-His-brace proteins PfCopC and LPMOs through classical biochemical characterization of the proteins and the absolute control over individual components, most importantly oxygen accessibility determining the oxidation state of co-substrates.