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-beta-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.

FIG. 1

Purification of TPS, TPP, and the fusion enzyme TPSP. (A) Construct for recombinant TPSP expression. The recombinant DNA for the TPS-TPP fusion enzyme (TPSP) was inserted into pRSETB. The N terminus of the hybrid enzyme was fused to a hexahistidine peptide (His tag), shown as a solid box. The predicted amino acid sequences of the fusion boundaries are shown. The N- and C-terminal sequences of TPS are underlined, whereas the N-terminal sequence of TPP is shown in bold. (B) SDS-PAGE of the purified proteins TPP, TPS, and TPSP. The recombinant enzymes were purified by Ni²⁺-NTA column and Mono Q 10/20 column chromatography as described in Materials and Methods. The purified proteins were analyzed by SDS–12.5% PAGE and stained with Coomassie brilliant blue. Lane M, protein size markers. Lanes 1 to 3, 2 μg of purified TPP, TPS, and TPSP, respectively. The numbers on the left indicate the sizes of the markers (in kilodaltons).

FIG. 2

Analysis of the reaction products of the fusion enzyme TPSP. The indicated purified recombinant enzymes (10 pmol each) were incubated for 60 min at 30°C in 100 μl of assay buffer containing 7.5 mM UDPG and 15 mM G6P or 10 mM T6P as substrates, and the reaction products were analyzed. (A) Silica gel TLC chromatogram. Lanes 1 to 5, 2 μg of standard glucose (G), UDPG, G6P, T6P, and trehalose (T), respectively. Lanes 6 to 9, 3 μl of the reaction products of TPS with G6P and UDPG, TPP with T6P, the TPS/TPP equimolar mixture with G6P and UDPG, and the TPSP fusion enzyme with G6P and UDPG, respectively. The identity of each spot is indicated on the left. (B) HPIC chromatogram. (a) A 52.5-ng amount of standard trehalose. (b and c) Control and reaction products of TPS/TPP mixture from G6P and UDPG after 0 and 60 min of incubation, respectively. (d) Reaction products of TPSP with G6P and UDPG after a 60-min incubation.

FIG. 3

Time course for trehalose production from UDPG and G6P by the recombinant enzyme. Ten picomoles of TPSP (●) or the TPS/TPP equimolar mixture (○) was incubated at 30°C in a final volume of 100 μl of assay buffer containing 7.5 mM UDPG and 15 mM G6P. Aliquots (10 μl) were withdrawn at the indicated times and quantified by HPIC.

FIG. 4

Lineweaver-Burk plots for trehalose synthesis catalyzed by TPSP or TPS/TPP in the presence of UDPG and G6P: effect of UDPG and G6P concentrations on enzyme activity. (A) Double-reciprocal plot of the initial velocity at various concentrations of UDPG against 30 mM G6P. Reactions involving the TPSP fusion enzyme (●) and TPS/TPP mixture (○) are shown. (B) Double-reciprocal plot of initial velocity at various concentrations of G6P against 15 mM UDPG. (C) Double-reciprocal plot of the initial velocity at various concentrations of T6P. Each point represents the mean of three determinations. Linear least-squares analyses were used to determine the slopes and intercepts in the double-reciprocal plots. Ten picomoles of TPSP, TPS, or TPP was used, and the reaction velocity (v) is expressed as nanomoles of trehalose formed per minute.

FIG. 5

Trehalose synthesis activity of TPSP and TPS/TPP. (A) Temperature dependence. Ten picomoles of TPSP (●) or the TPS/TPP equimolar mixture (○) was incubated for 60 min at various temperatures between 10 and 60°C in the standard assay mixture. After boiling for 3 min at 100°C, trehalose was analyzed as described in Materials and Methods. (B) Heat stability. One hundred picomoles of TPSP (●) or the TPS/TPP equimolar mixture (○) was incubated for the indicated lengths of time at 50°C in a final volume of 100 μl of a reaction mixture containing 33 mM Tris-HCl (pH 7.4) and 2.5 mM MgCl₂. (C) Heat stability of T6P phosphatase activity. One hundred picomoles of TPSP (●) or TPP (○) was incubated for the indicated lengths of time at 50°C in a final volume of 100 μl of a reaction mixture containing 33 mM Tris-HCl (pH 7.4) and 2.5 mM MgCl₂. After quenching on ice for 5 min, 7.5 mM UDPG and 15 mM G6P (B) or 10 mM T6P (C) was added to the reaction mixture. The remaining activity is expressed as a percentage of the original activity.

FIG. 6

In vivo synthesis of trehalose by overexpression of the bifunctional fusion enzyme TPSP. E. coli harboring plasmid pRSETB, pRTPS, pRTPP, or pRTPSP was induced with 0.5 mM IPTG for 3 h and lysed by sonication. (A) Each supernatant (5 μl) was spotted onto a TLC plate and analyzed as described in Materials and Methods. Lane 1, glucose; lane 2, trehalose; lane 3, pRSETB; lane 4, pRTPS; lane 5, pRTPP; lane 6, pRTPSP. (B) HPIC chromatogram. Trehalose, 110 ng of standard trehalose; before IPTG, sample before IPTG treatment; and IPTG (3 h), sample after IPTG treatment for 3 h. Five microliters of the cultured cells was analyzed. (C) Supernatants were analyzed by HPIC. Symbols are pRTPSP (●) and pRTPSP, no induction (○).