Mode of action and catalytic mechanisms of alginatemodifying enzymes Insights from the mannuronan C-5 epimerases and a bifunctional epimerase/lyase from Azotobacter vinelandii
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Enzymes are biological catalysts that work efficiently and specifically to make life possible. Among their countless tasks in living organisms, they are responsible for the diversity of material properties found in nature. If we understand how they function, it would open up immense possibilities for using them to produce materials and products from natural biomass. Alginate is a product derived from seaweed, with numerous applications due to its physical properties of viscosity and gel-formation. The properties are tuned from the composition of the two monomers β-d-mannuronic acid (M) and α-l-guluronic acid (G), found in block structures of M-blocks, G-blocks and alternating MG-blocks. G-residues are created from M-residues in these non-random patterns by the mannuronan C-5 epimerases (alginate epimerases). These enzymes are of interest to alginate producing industries, as they offer a way to tailor the alginate composition to a specific use. The thesis is concerned with understanding the molecular mechanisms behind the mode of action of the bacterial alginate epimerases AlgE from the soil bacterium Azotobacter vinelandii. They produce slightly different epimerization block patterns, and one of them has a dual alginate lyase/epimerase activity where it cleaves alginate chains through a β-elimination mechanism. Combined experimental and computational methodology was used to elucidate how charged residues are involved in substrate binding and processivity in AlgE4, and the direction of binding and movement was modelled. NMR and ITC experiments found that the balance between processivity and substrate binding is fine-tuned through the interplay between positively and negatively charged residues. Flexible loops surrounding the binding groove might help in attachment to the substrate during processive movement. Through a mutational investigation, residue 307 located in one of these loops was found to be essential for the epimerization pattern. The product pattern of the bifunctional AlgE7 was studied using NMR, unravelling a complex interplay between the two activities and effects on activity from changing concentrations of NaCl and Ca2+. Calcium has a structural role in the enzymes, and a combined computational and experimental study of the active module of AlgE6 implies that it might also have at least an indirect role in catalysis. The catalytic mechanism of AlgE7 was further studied, and roles of active site residues were proposed. H154 is most likely the catalytic base in epimerizations and cleavage of M-residues, and Y149 is most likely the catalytic acid. R148, found in the bifunctional AlgEs in place of G148 found in AlgEs with only epimerase activity, was essential for the lyase activity. Its proposed role is in affecting Y149 to donate a proton either to the opposite face of the sugar ring, in epimerizations, or to the glycosidic bond, in chain cleavage. Finally, time-resolved NMR was used to measure and model kinetics of AlgE4, and product inhibition was affirmed with ITC. The investigations of the mechanisms behind the AlgEs mode of action presented aid in understanding how they create their distinct product patterns. This is important for rational design of enzymes with new or improved functionalities for various needs. In addition, this work has shone new lights on an unusual system of polymer active enzymes. Although being carbohydrate-active, the epimerases are not classified in the CAZy-database. Their inclusion in the CAZypedia lexicon presented in the thesis might be a first step towards classifying these enzymes in a broader context.