Enzymatic synthesis of UDP-galactofuranose and an assay for UDP-galactopyranose mutase based on high-performance liquid chromatography.
A method to prepare UDP-galactofuranose (UDP-Galf) free of UDP-galactopyranose (UDP-Galp) is described. The UDP-Galf is synthesized enzymatically from UDP-Galp using the enzyme UDP-galactopyranose mutase. Treatment of UDP-Galp with the enzyme yields an equilibrium mixture of UDP-Galp and UDP-Galf in which UDP-Galf is approximately 7%. In spite of its low yield, the UDP-Galf is readily purified from starting UDP-Galp using a Dionex PA-100 ion exchange HPLC column. The purified UDP-Galf was characterized by chemical degradations, by electrospray mass spectrometry, and by several nuclear magnetic resonance techniques. In addition, an HPLC assay for the enzyme UDP-galactopyranose mutase is presented that requires 0.5 microgram of UDP-Galf per assay and can be used for both qualitative and quantitative measurements of the enzyme activity. These procedures should thus aid in the characterization of the enzymes involved in galactofuranosyl biosynthesis for the cell walls of Mycobacteria, for the lipophosphoglycan of Leishmania, and for other microorganisms where galactofuranosyl residues are found.
Lee
R
R
Department of Microbiology, Colorado State University, Fort Collins 80523-1677, USA.
Monsey
D
D
Weston
A
A
Duncan
K
K
Rithner
C
C
McNeil
M
M
D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005690:Galactose / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019751:Intramolecular Transferases; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013056:Spectrophotometry, Ultraviolet; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C104136:uridine diphosphate galactofuranose; D014530:Uridine Diphosphate; D002238:Carbohydrate Epimerases; D019751:Intramolecular Transferases; C490824:Glf protein, E coli; C098051:UDP-galactopyranose mutase; D005690:Galactose