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Applied and Environmental Microbiology, June 2000, p. 2484-2490, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Hak Soo Seo,1 Yeon Jong Koo,1 Jae Yun Lim,1 Jong Tae Song,1 Chung Ho Kim,2 Ju Kon Kim,3 Jong Seob Lee,4 and Yang Do Choi1,*

Graduate School of Agricultural Biotechnology, Seoul National University, Suwon 441-744,1 Department of Food and Nutrition, Seowon University, Cheongju 361-742,2 Department of Biological Science, MyongJi University, Yongin 449-728,3 and Graduate School of Biological Sciences, Seoul National University, Seoul 151-742,4 Korea

Received 22 December 1999/Accepted 20 March 2000

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 Km 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 kcat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (kcat/Km). The Km and kcat 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.

* Corresponding author. Mailing address: Graduate School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea. Phone: 82-331-290-2407. Fax: 82-331-291-7011. E-mail: choiyngd{at}snu.ac.kr.

Applied and Environmental Microbiology, June 2000, p. 2484-2490, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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