Abstract
Ectoine biosynthetic genes (ectABC) are widely distributed in bacteria. Microorganisms that carry them make copious amounts of ectoine as a cell protectant in response to high-osmolarity challenges. Ectoine synthase (EctC; EC 4.2.1.108) is the key enzyme for the production of this compatible solute and mediates the last step of ectoine biosynthesis. It catalyzes the ring closure of the cyclic ectoine molecule. A codon-optimized version of ectC from Sphingopyxis alaskensis (Sa) was used for overproduction of SaEctC protein carrying a Strep-tag II peptide at its carboxy-terminus. The recombinant SaEctC-Strep-tag II protein was purified to near-homogeneity from Escherichia coli cell extracts by affinity chromatography. Size-exclusion chromatography revealed that it is a dimer in solution. The SaEctC-Strep-tag II protein was crystallized using the sitting-drop vapour-diffusion method and crystals that diffracted to 1.0 Å resolution were obtained.