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Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.
Eur J Biochem 2002; 269(24):6091-100EJ

Abstract

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 approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli (approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 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, i.e. 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 glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.

Authors+Show Affiliations

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

12473104

Citation

Pan, Y T., et al. "Trehalose-phosphate Synthase of Mycobacterium Tuberculosis. Cloning, Expression and Properties of the Recombinant Enzyme." European Journal of Biochemistry, vol. 269, no. 24, 2002, pp. 6091-100.
Pan YT, Carroll JD, Elbein AD. Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme. Eur J Biochem. 2002;269(24):6091-100.
Pan, Y. T., Carroll, J. D., & Elbein, A. D. (2002). Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme. European Journal of Biochemistry, 269(24), pp. 6091-100.
Pan YT, Carroll JD, Elbein AD. Trehalose-phosphate Synthase of Mycobacterium Tuberculosis. Cloning, Expression and Properties of the Recombinant Enzyme. Eur J Biochem. 2002;269(24):6091-100. PubMed PMID: 12473104.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme. AU - Pan,Y T, AU - Carroll,J D, AU - Elbein,A D, PY - 2002/12/11/pubmed PY - 2003/3/7/medline PY - 2002/12/11/entrez SP - 6091 EP - 100 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 269 IS - 24 N2 - 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 approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli (approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 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, i.e. 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 glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/12473104/Trehalose_phosphate_synthase_of_Mycobacterium_tuberculosis__Cloning_expression_and_properties_of_the_recombinant_enzyme_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=2002&volume=269&issue=24&spage=6091 DB - PRIME DP - Unbound Medicine ER -