Mechanistic analysis of trehalose synthase from Mycobacterium smegmatis

J Biol Chem. 2011 Oct 14;286(41):35601-35609. doi: 10.1074/jbc.M111.280362. Epub 2011 Aug 12.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Deuterium Exchange Measurement
  • Glucosyltransferases / chemistry*
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism
  • Glycoside Hydrolases / chemistry
  • Glycoside Hydrolases / genetics
  • Glycoside Hydrolases / metabolism
  • Kinetics
  • Mycobacterium smegmatis / enzymology*
  • Mycobacterium smegmatis / genetics
  • Substrate Specificity


  • Bacterial Proteins
  • Glucosyltransferases
  • trehalose synthase
  • Glycoside Hydrolases