Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus.
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 approximately 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 microM 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 microM and 90 +/- 30 microM, respectively. Functional and structural differences between the bacterial and eukaryotic UGMs are discussed.
Oppenheimer
Michelle
M
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
Poulin
Myles B
MB
Lowary
Todd L
TL
Helm
Richard F
RF
Sobrado
Pablo
P
D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D008024:Ligands; D011994:Recombinant Proteins; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase