Evaluation and Comparison of vs Mammalian 6-O-Phospho-Kinases: Substrate Specificity and Applications.

Sensing of peptidoglycan fragments is essential for inducing downstream signaling in both mammalian and fungal systems. The hexokinases NagK and Hxk1 are crucial enzymes for the phosphorylation of peptidoglycan molecules in order to activate specific cellular responses; however, biochemical characterization of their enzymatic specificity and efficiency has yet to be investigated in depth. Here a mass spectrometry enzymatic screen was implemented to assess substrate specificity, and an ATP coupled assay provided the quantitative kinetic profiles of these two homologous, eukaryotic enzymes. The data show, that while homologous, NagK and Hxk1 have vastly different substrate profiles. NagK accepts a variety of different peptidoglycan-based substrates albeit with reduced efficiency but are still valuable as a tool in large scale chemoenzymatic settings. Conversely, Hxk1 has a smaller substrate scope but can turnover these alternative substrates at similar levels to its natural substrate. These results allow for deeper understanding into the biosynthetic machinery responsible for essential cellular processes including UDP-GlcNAc regulation and immune recognition events in the cell.