Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus

Abstract

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose, which is an important cell wall component in Aspergillus fumigatus and other pathogenic microbes. A. fumigatus UGM ( AfUGM) was expressed in Escherichia coli and purified to homogeneity. The enzyme was shown to function as a homotetramer by size-exclusion chromatography and to contain ∼50% of the flavin in the active reduced form. A k cat value of 72 ± 4 s −1 and a K M value of 110 ± 15 μM were determined with UDP-galactofuranose as substrate. In the oxidized state, AfUGM does not bind UDP-galactopyranose, while UDP and UDP-glucose bind with K d values of 33 ± 9 μM and 90 ± 30 μM, respectively. Functional and structural differences between the bacterial and eukaryotic UGMs are discussed.