Construction of an artificial bifunctional enzyme, .beta.-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling

Ljungcrantz, Peter; Carlsson, Helén; Mo, Månsson; Buckel, Peter; Mosbach, Klaus; Bülow, Leif

doi:10.1021/bi00448a016

Cited by 80 publications

(47 citation statements)

References 24 publications

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“…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). Unlike these reports, due to the monomeric nature of the ␣1,3GalT, both fusion proteins f1 and f2 in the present study exist as homodimers.…”

Section: Discussioncontrasting

confidence: 48%

“…Significantly, the reaction rates in producing Gal␣1,3Lac from UDP-Glc and lactose are increased 4-and 1.5-fold by f1 and f2, respectively, in comparison with the native enzymes. This result can be explained by the substrate channeling effect (21), indicating that the UDP-Gal produced by the epimerase is captured faster by the transferase moiety in either f1 or f2 than that in the system of native enzymes. Furthermore, the higher activity of f1 over f2 suggests that the active sites for the epimerase and ␣1,3-galactosyltransferase part are in a more optimal configuration in f1.…”

Section: Discussionmentioning

confidence: 94%

“…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).…”

mentioning

confidence: 99%

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

Chen

Liu

Wang

et al. 2000

Journal of Biological Chemistry

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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 His 6 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 ␣1,3-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 ␣1,3-galactosyltransferase from UDP-galactose to UDP-glucose and decreased the cost for the synthesis of biomedically important Gal␣1, 3Gal-terminated oligosaccharides by more than 40-fold. For enzymatic synthesis of Gal␣1,3Gal␤1,4Glc 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 ␣1,3-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.Oligosaccharides are attractive targets for the development of new pharmaceuticals because of their important roles in cell recognition, cell signaling, and other biological processes (1, 2). ␣-Gal 1 epitopes (Gal␣1,3Gal-terminated oligosaccharide sequences including di-, tri-, and pentasaccharides) have drawn increasing attention since it was discovered that the interaction of preexisting natural antibodies in human serum with this specific xenoactive oligosaccharide sequence on animal cells is the main cause of hyperacute rejection in xenotransplantation (3). ␣-Gal epitopes exist as glycolipids or glycoproteins on the cell surface of mammals other than humans, apes, and Old World Monkeys (4, 5). The unique enzyme responsible for the formation of the terminal glycoside bond in nature is UDP-Gal:Gal␤1,4GlcNHAc ␣1,3-galactosyltransferase (␣1,3GalT), a protein that is absent in humans due to mutational inactivation of the gene (6, 7). In contrast, humans produce a large amount of anti-Gal antibodies including IgG, IgM, and IgA isotypes (8).The discovery of the interaction of anti-Gal and ␣-Gal epitopes has led to experimental attempts to overcome hyperacute rejection by either depleting the recip...

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Section: Discussioncontrasting

confidence: 48%

Section: Discussionmentioning

confidence: 94%

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

Chen

Liu

Wang

et al. 2000

Journal of Biological Chemistry

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“…no intrinsic differences in the structures of each enzyme moiety in the fusion enzyme and the individual enzymes, even though TPSP was slightly more stable than the TPS/TPP mixture. Still, some studies showed that the fusion enzyme was more stable against thermal denaturation than the individual enzymes were (16,30).…”

Section: Discussionmentioning

confidence: 99%

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

Seo

Koo

Lim

et al. 2000

Appl Environ Microbiol

100

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To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The K m values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the k cat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5-to 4.0-fold increase in the catalytic efficiency (k cat /K m ). The K m and k cat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-␤-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.The nonreducing disaccharide trehalose [␣-D-glucopyranosyl-(131)-␣-D-glucopyranose] has high water-holding activities, which maintain the fluidity of membranes under dry conditions (25). It also stabilizes enzymes, foods, cosmetics, and pharmaceuticals at high temperatures (8,9,38). Due to its desirable physical and chemical characteristics, commercial production of trehalose is anticipated. Escherichia coli synthesizes trehalose when exposed to high osmolarity (12,20,39,41). In E. coli, trehalose is synthesized by two separate enzymes, trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP), encoded by the genes otsA and otsB, respectively (15, 21). This is different from Saccharomyces cerevisiae, in which trehalose is synthesized by a large multisubunit complex with the catalytic activities of both TPS and TPP (6, 31, 43).Overexpression of TPS and TPP might be one way to produce trehalose. We assumed that the physical proximity of two enzymes catalyzing sequential reactions might increase the reaction rate by facilitating transfer of the reaction intermediate when they are present in a complex. A variety of techniques have been applied to better understand the proximity effect of enzymes catalyzing sequential reactions, including cross-linking and coimmobilization (23,32,34). In many of these cases,...

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Kim

Kwon

Park

et al. 2000

Appl Environ Microbiol

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A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. Recombinant BvMTS, BvMTH, and fusion gene BvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of ␣-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.

show abstract

Construction of an artificial bifunctional enzyme, .beta.-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling

Cited by 80 publications

References 24 publications

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

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

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

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

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