Publications

• Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

  Pan Y.T., Koroth Edavana V., Jourdian W.J., Edmondson R., Carroll J.D., Pastuszak I., Elbein A.D.

  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 ... >> More

  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. << Less

  Eur. J. Biochem. 271:4259-4269(2004) [PubMed] [EuropePMC]

• Mechanistic analysis of trehalose synthase from mycobacterium smegmatis.

  Zhang R., Pan Y.T., He S., Lam M., Brayer G.D., Elbein A.D., Withers S.G.

  Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmac ... >> More

  Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. TreS catalyzes the hydrolytic cleavage of α-aryl glucosides as well as α-glucosyl fluoride, thereby allowing facile, continuous assays. Reaction of TreS with 5-fluoroglycosyl fluorides results in the trapping of a covalent glycosyl-enzyme intermediate consistent with TreS being a member of the retaining glycoside hydrolase family 13 enzyme family, thus likely following a two-step, double displacement mechanism. This trapped intermediate was subjected to protease digestion followed by LC-MS/MS analysis, and Asp(230) was thereby identified as the catalytic nucleophile. The isomerization reaction was shown to be an intramolecular process by demonstration of the inability of TreS to incorporate isotope-labeled exogenous glucose into maltose or trehalose consistent with previous studies on other TreS enzymes. The absence of a secondary deuterium kinetic isotope effect and the general independence of k(cat) upon leaving group ability both point to a rate-determining conformational change, likely the opening and closing of the enzyme active site. << Less

  J. Biol. Chem. 286:35601-35609(2011) [PubMed] [EuropePMC]

• Purification and properties of a novel enzyme, trehalose synthase, from Pimelobacter sp. R48.

  Nishimoto T., Nakano M., Nakada T., Chaen H., Fukuda S., Sugimoto T., Kurimoto M., Tsujisaka Y.

  A novel enzyme, trehalose synthase, was purified from a cell-free extract of Pimelobacter sp. R48 to an electrophoretically homogeneous state by successive chromatographies on DEAE-Toyopearl 650, Butyl-Toyopearl 650, and Mono Q HR5/5 columns. The molecular weight of the enzyme was estimated to be ... >> More

  A novel enzyme, trehalose synthase, was purified from a cell-free extract of Pimelobacter sp. R48 to an electrophoretically homogeneous state by successive chromatographies on DEAE-Toyopearl 650, Butyl-Toyopearl 650, and Mono Q HR5/5 columns. The molecular weight of the enzyme was estimated to be 62,000 by SDS-polyacrylamide gel electrophoresis, and the enzyme had a pI of 4.6 by gel isoelectrofocusing. The enzyme catalyzed the conversion of maltose into trehalose by intramolecular transglucosylation. The enzyme also converted into maltose but was inactive on other saccharides. The N-terminal amino acid of the enzyme was serine. The optimum pH and temperature were pH7.5 and 20 degrees C, respectively. The enzyme was stable in the range of pH 6.0-9.0 and up to 30 degrees C for 60 min. The rate of conversion of maltose into trehalose was independent of the maltose concentration. The maximum yield of trehalose from maltose were 81.8%, 80.9%, and 76.7% at 5, 15, and 25 degrees C, respectively. The activity was inhibited by Cu2+, Hg2+, Ni2+, Zn2+, and Tris. << Less

  Biosci Biotechnol Biochem 60:640-644(1996) [PubMed] [EuropePMC]