Structural framework of fructosyl transfer in Bacillus subtilis levansucrase

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Fructans, polymers of beta-D-fructofuranose, serve as primary carbohydrate reserves in many plants and bacteria.
• These fructans, like microbial levan (beta(2,6)-linked), are believed to enhance plant tolerance to environmental stresses such as drought and frost.
• Levansucrase enzymes synthesize levan directly from sucrose, exhibiting both hydrolysis and polymerization activities.

Purpose of the Study:

• To elucidate the structural basis of Bacillus subtilis levansucrase activity.
• To identify key amino acid residues involved in the catalytic mechanism of levan synthesis.

Main Methods:

• X-ray crystallography was employed to determine the enzyme's structure at high resolution (1.5 A).
• The structure of a catalytically inactive mutant (E342A) bound to sucrose was also determined (2.1 A).
• Site-directed mutagenesis was used to probe the function of specific residues.

Main Results:

• The crystal structure revealed a unique five-fold beta-propeller fold with a negatively charged central pocket.
• A critical residue, Arg360, essential for polymerase activity, is located near this pocket.
• Mutagenesis and structural data indicate that three conserved acidic residues (Asp86, Glu342, Asp247) within the central pocket are crucial for catalysis.

Conclusions:

• The structure of Bacillus subtilis levansucrase provides insights into the mechanism of levan synthesis.
• Specific acidic residues in the central pocket likely act as a catalytic triad, facilitating sucrose hydrolysis and levan polymerization.
• Understanding this enzyme's structure-function relationship can inform strategies for manipulating fructan production.