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Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.
Eur J Biochem 2004; 271(21):4259-69EJ

Abstract

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.

Authors+Show Affiliations

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

Pub Type(s)

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

Language

eng

PubMed ID

15511231

Citation

Pan, Yuan T., et al. "Trehalose Synthase of Mycobacterium Smegmatis: Purification, Cloning, Expression, and Properties of the Enzyme." European Journal of Biochemistry, vol. 271, no. 21, 2004, pp. 4259-69.
Pan YT, Koroth Edavana V, Jourdian WJ, et al. Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme. Eur J Biochem. 2004;271(21):4259-69.
Pan, Y. T., Koroth Edavana, V., Jourdian, W. J., Edmondson, R., Carroll, J. D., Pastuszak, I., & Elbein, A. D. (2004). Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme. European Journal of Biochemistry, 271(21), pp. 4259-69.
Pan YT, et al. Trehalose Synthase of Mycobacterium Smegmatis: Purification, Cloning, Expression, and Properties of the Enzyme. Eur J Biochem. 2004;271(21):4259-69. PubMed PMID: 15511231.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme. AU - Pan,Yuan T, AU - Koroth Edavana,Vineetha, AU - Jourdian,William J, AU - Edmondson,Rick, AU - Carroll,J David, AU - Pastuszak,Irena, AU - Elbein,Alan D, PY - 2004/10/30/pubmed PY - 2004/12/18/medline PY - 2004/10/30/entrez SP - 4259 EP - 69 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 271 IS - 21 N2 - Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/15511231/Trehalose_synthase_of_Mycobacterium_smegmatis:_purification_cloning_expression_and_properties_of_the_enzyme_ L2 - https://doi.org/10.1111/j.1432-1033.2004.04365.x DB - PRIME DP - Unbound Medicine ER -