title: Overproduction, crystallization and X-ray diffraction data analysis of ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis.
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.
authors.affiliation: Crystal Farm and X-ray Facility, Heinrich-Heine-University, Universitaetsstrasse 1, 40225 Düsseldorf, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kobus
authors.affiliation: Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, Karl-von-Frisch Strasse 8, 35043 Marburg, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Widderich
authors.affiliation: Crystal Farm and X-ray Facility, Heinrich-Heine-University, Universitaetsstrasse 1, 40225 Düsseldorf, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Hoeppner
authors.affiliation: Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, Karl-von-Frisch Strasse 8, 35043 Marburg, Germany.
authors.forename: Erhard
authors.initials: E
authors.lastname: Bremer
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University, Universitaetsstrasse 1, 40225 Düsseldorf, Germany.
authors.forename: Sander H J
authors.initials: SH
authors.lastname: Smits
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D059001:Aquatic Organisms; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003080:Cold Temperature; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D042301:Sphingomonadaceae / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D006836:Hydro-Lyases; C106231:ectoine synthase
keywords: X-ray analysis; chemical chaperone; compatible solute; cupin; ectoine synthesis; enzyme; osmostress protectant