Nick Smith MSc Student, diCenzo Lab Characterizing bifunctional sugar-processing enzymes from thermophilic bacteria Nucleotide sugars are the donor substrates for glycosyltransferase enzymes, which are widely studied throughout biochemistry, medicine, and glycobiology. However, nucleotide sugars are prohibitively expensive, prompting the development of in-lab chemoenzymatic synthesis protocols to circumvent this cost. One such example is the usage of the bifunctional Fucokinase/GDP-fucose pyrophosphorylase enzyme from Bacteroides fragilis (BfFKP) to catalyze the production of the costly compound GDP-Fucose. The BfFKP enzyme contains a C-terminal Fucokinase domain and an N-terminal GDP-Fucose pyrophosphorylase domain that catalyze sequential steps in the GDP-Fucose synthesis pathway. As a result, this bifunctional protein is used in “one-pot” syntheses, resulting in appreciable amounts of GDP-Fucose for a fraction of the commercial cost. Since the discovery of the BfFKP enzyme, studies have mainly focused on using this enzyme rather than sourcing novel FKP proteins, despite an enormous sequence space of putative FKP orthologs. To address this lack of knowledge, a sequence similarity network (SSN) was used to identify putative bifunctional FKP enzymes. To test the hypothesis that FKP enzymes from thermophilic organisms would exhibit altered properties and/or activities, an uncharacterized FKP sequence from the thermophile Thermophagus xiamenesis (i.e., TxFKP) was selected. I recombinantly overexpressed both the TxFKP and BfFKP enzymes in Escherichia coli and purified the proteins using Immobilized Metal Ion Affinity Chromatography. To compare thermostability between the two enzymes, I used Thermal Shift Assays to approximate their melting temperatures (Tm). Unexpectedly, both proteins demonstrated biphasic melt curves, suggesting that the enzymatic domains are modular enough to unfold at separate temperatures in vitro. In addition, I performed kinetic analyses of the Fucokinase domains of both enzymes, revealing key differences and similarities between the two. These results will shed light on the variance of properties within a fascinating group of biocatalytic tools.
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