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

Abstract

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,...

A bifunctional TPS–TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis

“…This fusion protein yielded an active bifunctional TPS and TPP enzyme with trehalose biosynthetic capacity and restored stress tolerance when expressed in yeast. A similar construct has been reported for the fusion of the E. coli otsA and otsB genes encoding TPS and TPP, respectively, which has both enzyme activities and synthesizes trehalose when expressed in bacteria (Seo et al 2000).…”

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

“…, respectively) are very high when compared to the values of TPS and TPP from E. coli (Seo et al 2000). Moreover, the Thermus enzymes have also lower K m values, meaning that these enzymes have higher catalytic efficiency, when compared to that from E. coli.…”

“…examined have high thermo- Relative activity(%) stability, as would be expected (Koyama and Furukawa 1990;Wang et al 2003). The TPP from the mesophilic M. smegmatis, on the other hand, was completely inactivated after incubation for 0.5 min at 100°C (Klutts et al 2003), and a mixture of TPS/TPP from E. coli retained only 3% of the original activity after incubation at 50°C for 30 min (Seo et al 2000). The enhanced stability of enzymes at high temperature is the result of differences in specific amino acid composition (Vieille et al 1996).…”

Evolution of Sucrose Synthesis

“…The C-terminal region of the Synechocystis SPS shows 42% identity to the Synechocystis SPP (Lunn et al, 2000) but lacks several of the conserved, active site residues and does not have SPP activity (Lunn et al, 1999). Seo et al (2000) expressed a fusion protein of the E. coli trehalose-P-synthase (TPS) and trehalose-phosphatase (TPP) and found that the chimeric protein had both TPS and TPP activities. TPS and TPP are functionally and structurally related to SPS and SPP, leading to the question of whether a single polypeptide can have both SPS and SPP activities.…”