Functional characterization of human phosphomannomutases by 31P-NMR

PMM2-CDG is caused by mutations in the gene PMM2 encoding phosphomannomutase2. Yet a paralogous enzyme exists in humans. PMM1 has an additional phosphatase activity and is able to hydrolyze glucose and mannose 1,6 bisphosphate. This latter activity is enhanced by increased concentrations of Inosine monophosphate (IMP). The specific role of the two enzymes in vivo is not fully understood and in particular the effect of PMM1 mutations on hypomorphic PMM2 has not been evaluated yet and IMP could play an important part. For this reason we believe that it is worth elucidating the functioning of PMM1. The first step towards protein N- or C-glycosylation requires the isomerization of mannose 6-phosphate into mannose 1-phosphate. In vitro however PMM1 and PMM2 are assayed measuring their phosphomutase activity in the opposite direction. Using 31P-NMR spectroscopy we tested both enzymes in the physiologically relevant direction, as well as the phosphatase activity of PMM1 in the presence or in the absence of IMP. We identified the residues which are responsible for the sensitivity of PMM1 to IMP and we modelled the interaction with the nucleotide using a docking program that consents full flexibility of the protein as well as the ligand.