Abstract
Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC ) and alpha1, 3-galactosyltransferase (EC ) with an N-terminal His(6) tag and an intervening three-glycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3' terminus (f1) or to the 5' terminus (f2) of a truncated bovine alpha1, 3-galactosyltransferase gene, respectively. Both fusion proteins were expressed in cell lysate as active, soluble forms as well as in inclusion bodies as improperly folded proteins. Both f1 and f2 were determined to be homodimers, based on a single band observed at about 67 kDa in SDS-polyacrylamide gel electrophoresis and on a single peak with a molecular mass around 140 kDa determined by gel filtration chromatography for each of the enzymes. Without altering the acceptor specificity of the transferase, the fusion with the epimerase changed the donor requirement of alpha1, 3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Galalpha1,3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Galalpha1,3Galbeta1,4Glc from UDP-glucose and lactose, the genetically fused enzymes f1 and f2 exhibited kinetic advantages with overall reaction rates that were 300 and 50%, respectively, higher than that of the system containing equal amounts of epimerase and galactosyltransferase. These results indicated that the active sites of the epimerase and the transferase in fusion enzymes were in proximity. The kinetic parameters suggested a random mechanism for the substrate binding of the alpha1, 3-galactosyltransferase. This work demonstrated a general approach that fusion of a glycosyltransferase with an epimerase can change the required but expensive sugar nucleotide to a less expensive one.
Highlights
Two fusion enzymes consisting of uridine diphosphogalactose 4-epimerase (UDP-galactose 4-epimerase, EC 5.1.3.2) and ␣1,3-galactosyltransferase (EC 2.4.1.151) with an N-terminal His6 tag and an intervening threeglycine linker were constructed by in-frame fusion of the Escherichia coli galE gene either to the 3 terminus (f1) or to the 5 terminus (f2) of a truncated bovine ␣1,3galactosyltransferase gene, respectively
We showed that a simpler alternative was a two-enzyme system in which UDP-galactose 4-epimerase (GalE) converted relatively inexpensive sugar nucleotide UDPGlc to UDP-Gal, and ␣1,3-galactosyltransferase carried out the subsequent glycosylation reaction [19]
NdeI and BamHI restriction sites facilitated the insertion of the ␣1,3GalT gene into the multiple restriction sites in vector pET15b, while SpeI and SalI were for the introduction of the galE gene
Summary
Chemicals and Reagents—Plasmid vector pET15b was purchased from Novagen Inc., Madison, WI. The column was eluted with the same buffer, and fractions containing fusion protein activity were pooled and concentrated in a Centricon 3 concentrator To obtain the active fusion proteins from the inclusion bodies, the purification and refolding procedures were carried out as described in. One unit of fusion enzyme activity is defined as the amount of enzyme that catalyzes the transfer of 1 mol of galactose from UDP-Glc to lactose/min at 37 °C. UDP-[6-3H]Glc (0.3 mM) and varying concentrations of lactose (7, 8, 10, 12.5, 17, 25, and 50 mM) at 37 °C for 30 min, in which the formation of the product [6-3H]Gal␣1,3Lac was measured by scintillation counting. Enzymatic activity of the epimerase moiety (UDP-Glc 3 UDP-Gal reaction) in the fusion enzymes was obtained with different concentrations of UDP-[6-3H]Glc (1, 1.14, 1.4, 1.6, 2, 2.6, and 4 mM) at 24 °C for. The concentrations of all enzymes were determined by the Lowry method [28] and adjusted to 0.3 M for fusion enzymes f1 and f2 as well as for native GalE and ␣1,3GalT
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