TAILIEUCHUNG - Báo cáo Y học: Trehalose-phosphate synthase of Mycobacterium tuberculosis Cloning, expression and properties of the recombinant enzyme

The trehalose-phosphate synthase (TPS) ofMycobacterium smegmatiswas previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Basedon that sequence information, the gene for TPSwasidentified inthe Mycobacterium tuberculosis genome, and the gene was cloned and expressed inEscheri-chia coliwith a (His)6tag at the amino terminus. | Eur. J. Biochem. 269 6091-6100 2002 FEBS 2002 doi Trehalose-phosphate synthase of Mycobacterium tuberculosis Cloning expression and properties of the recombinant enzyme Y. T. Pan1 J. D. Carroll2 and A. D. Elbein1 1 Department of Biochemistry and Molecular Biology and 2Department of Microbiology and Immunology University of Arkansas for Medical Sciences Little Rock Arkansas USA The trehalose-phosphate synthase TPS of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information the gene for TPS was identified in the Mycobacterium tuberculosis genome and the gene was cloned and expressed in Escherichia coli with a His 6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme and was purified on a metal ion column to give a single band of w 58 kDa on SDS PAGE. Approximately mg of purified TPS were obtained from a 1-L culture of E. coll w g cell paste . The purified recombinant enzyme showed a single band of w 58 kDa on SDS PAGE but a molecular mass of w 220 kDa by gel filtration indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors . ADP-glucose CDP-glucose GDP-glucose TDP-glucose and UDP-glucose with ADP-glucose GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the .

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