Characterization of human and chicken PGM2 and PGM5

Abstract

The proteins of the α-D-phosphohexomutase superfamily are important in several metabolic processes in all kingdoms of life. Many of the superfamily members are, however, severely understudied. Two of the less studied human members are phosphoglucomutase 2 (PGM2) and phosphoglucomutase 5 (PGM5), which were selected for investigations in this project. The α-D-phosphohexomutase family members are responsible for the reversible intramolecular transfer of a phosphoryl group on phosphosugars. According to the literature, PGM5 has no proven enzymatic activity, however, due to a high degree of sequence identity with the well-studied PGM1, PGM5 appears to be promising for further investigations. In this thesis, human and chicken PGM2 and PGM5 solubility, expression and purification were explored. Purification of PGM2 was optimized and crystallization screening experiments were conducted on both variants. The expression and solubility of PGM5 were explored, and the addition of a soluble MBP-tag gave rise to soluble hPGM5. The proteins were purified with the use of affinity chromatography and size exclusion chromatography. Both proteins had preliminary phosphoglucomutase activity tests conducted on them. Members of the α-D-phosphohexomutases superfamily have been linked to genetic diseases in humans, and better understanding the function and structure of PGM2 and PGM5 are likely to be of interest medically. As no protein crystals were obtained, the structures were examined by building homology models. Homology modelling was applied to get theoretical models for the proteins when no 3D structures were obtained through the laboratory work. The models of the active site suggested explanations of the function of the proteins. The active site model of hPGM2 showed a possible for the substrate variation of the enzyme. Examining hPGM5 and comparing it to hPGM1 showed no evidence of PGM5 lacking enzymatic activity due to differences in the active site.