Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase

Abstract

A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E. coli gene galT to the 3'-terminus of the E. coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid. The fusion enzyme was expressed in E. coli and purified 24-fold to about 98% homogeneity by chromatography on hydroxylapatite and Q-Sepharose. On the basis of the comparison of the elution profile for enzyme activities upon gel permeation chromatography (Sephacryl S-400) with the molecular weight of 80,000 determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the fusion enzyme appears to exist in monomeric, dimeric, and tetrameric forms, all of which exhibit both enzymatic activities. The Km values of the fusion enzyme for substrates were similar to those for the corresponding native enzymes, except for UDP-glucose, but the kcat values were smaller than those for the native enzymes. The fusion enzyme shows kinetic advantages in that the initial velocity to produce glucose-1-P from UDP-galactose and galactose-1-P is about 20% faster than that for a mixture of equal activities of the separate enzymes.

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

“…It was previously shown that differences in the K m values of native enzymes and fusion proteins reflect the presence of specific interactions resulting from the fusion of the enzymes (5,28). A proximity effect or substrate channeling operates in a fusion enzyme and increases the overall catalytic activity of the reaction (4,13,14,26,27,33,35,42). In this process, the reaction product of the first enzyme is transferred directly to the active site of the second enzyme in the sequential enzyme complex or fusion enzyme, without diffusing freely in solution.…”

Changing the Donor Cofactor of Bovine α1,3-Galactosyltransferase by Fusion with UDP-galactose 4-Epimerase

“…A previous report about a fusion protein composed of UDPGal 4-epimerase and galactose-1-phosphate uridylyltransferase (GalT) with an intervening Ala 3 linker was shown to exist in three forms: a monomer, dimer, or tetramer (20). Another fusion protein of ␤-galactosidase and galactose dehydrogenase was also shown to exist as two major forms, consisting of four or six subunits, as well as other forms (21).…”

“…These fusion enzymes change the donor requirement of the ␣1,3GalT from UDP-Gal to UDP-Glc. Furthermore, the fused enzyme system that catalyzes a sequential reaction may also have a kinetic advantage over the mixture of two separated enzymes, since the product of one enzyme travels a shorter distance to be captured by the next enzyme (20,21).…”

Trehalose Synthesis by Sequential Reactions of Recombinant Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase from Brevibacterium helvolum

“…Such proximity allows a reaction intermediate to be directly transferred to the active site of the next enzyme in a sequential enzyme complex. By preventing serious diffusion of the intermediate, the reaction rates of whole enzymatic processes could be increased (2,13,22). Recently, we reported that a bifunctional fusion enzyme resulting from fusion between T6P synthase and trehalose 6-phosphatase of E. coli exhibited increased catalytic activity during trehalose synthesis (20).…”