Purification, crystallization, and X-ray crystallographic analysis of D-allulose epimerase from Leucobacter ruminantium

Purification, crystallization, and X-ray crystallographic analysis of D-allulose epimerase from Leucobacter ruminantium

D-allulose epimerase (DAE) catalyzes the C-3 epimerization reaction that converts d-fructose to d-allulose. The preliminary structural study on the annotated DAE from Leucobacter ruminantium (LrDAE) was performed to understand the interaction between enzyme and substrate or product, which may provide structural background to design mutants for higher production of d-allulose from d-fructose than the wild-type and other enzymes. For this, the LrDAE encoding gene was cloned and expressed in Escherichia coli . The purified LrDAE protein was crystallized from the precipitant composed of 0.3 M Magnesium acetate, 0.1 M Sodium citrate (pH 5.6), and 13% (w/v) polyethylene glycol 8000. Diffraction data was collected to 3.0 Å resolution. The crystal belongs to the primitive orthorhombic P212121 space group with unit-cell parameters a = 69.58 Å, b = 117.65 Å, c = 150.47 Å, and α = β = γ = 90°. There are four LrDAE molecules in the asymmetric unit.