TAILIEUCHUNG - Báo cáo khoa học: Trehalose synthase of Mycobacterium smegmatis Purification, cloning, expression, and properties of the enzyme

Trehalose synthase (TreS) catalyzes the reversible inter-conversion of trehalose (glucosyl-a,a-1,1-glucose) and maltose (glucosyl-a1-4-glucose). TreS was purified from the cytosol ofMycobacterium smegmatisto give a single protein band on SDS gels with a molecular mass of 68 kDa. However, active enzyme exhibited a molecular mass of 390 kDa by gel filtration suggesting that TreS is a hexa-mer of six identical subunits. | Eur. J. Biochem. 271 4259-4269 2004 FEBS 2004 doi Trehalose synthase of Mycobacterium smegmatis Purification cloning expression and properties of the enzyme Yuan T. Pan1. Vineetha Koroth Edavana1. William J. Jourdian2. Rick Edmondson3 J. David Carroll4 . . . Irena Pastuszak1 and Alan D. Elbein1 1 Department of Biochemistry and Molecular Biology and Department of Microbiology and Immunology University of Arkansas for Medical Sciences Little Rock AR USA 2Departments of Biological Chemistry and Internal Medicine University of Michigan Medical Center Ann Arbor MI USA 3National Center for Toxicological Research Jefferson AR USA Trehalose synthase TreS catalyzes the reversible interconversion of trehalose glucosyl-a a-1 1-glucose and maltose glucosyl-a1-4-glucose . TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of w 68 kDa. However active enzyme exhibited a molecular mass of w 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 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 or 2 mM only about 30 of the trehalose was converted to maltose in 12 h indicating that maltose is the preferred substrate. These incubations also produced up to 8-10 free glucose. The Km for maltose was w 10 mM whereas for .

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