Cloning, expression, purification, crystallization and preliminary X-ray analysis of EaLsc, a levansucrase from Erwinia amylovora

EaLsc, a levansucrase from E. amylovora, has been cloned, expressed, purified and crystallized. X-ray crystallographic analysis and data collection to 2.77 Å resolution have been carried out. The structure was solved by molecular replacement and refinement is in progress. The Gram-negative bacterium Erwinia amylovora is a destructive pathogen of Rosaceae. During infection, E. amylovora produces the exopolysaccharide levan, which contributes to the occlusion of plant vessels, causing the wilting of shoots. Levan is a fructose polymer that is synthesized by multifunctional enzymes called levansucrases. The levansucrase from E. amylovora (EaLsc) was heterologously expressed as a GST-fusion protein in Escherichia coli, purified and crystallized after tag removal. The protein crystallized in space group P2₁2₁2. X-ray diffraction data were acquired to 2.77 Å resolution. The structure of the enzyme was solved by molecular replacement. The asymmetric unit contains eight enzyme molecules, giving a solvent content of 58.74% and a Matthews coefficient of 2.98 ų Da^-1