Sis EctD protein. The S. alaskensis EctD protein was purified by affinity chromatography and its quaternary structure was then assessed by gel filtration analysis on a HiLoad 16/600 Superdex 200 pg column. The column was equilibrated and run inside a 20 mM TESbuffer containing 150 NaCl. A protein option [3 mg/ml] of carbonic anhydrase (from bovine erythrocytes) (29 kDa), albumin (from bovine serum) (66 kDa), and alcohol dehydrogenase (from Saccharomyces cerevisiae) (150 kDa) was applied as a normal. The calculated molecular mass of your S. alaskensis EctD protein with the attached Strep-tag-II affinity peptide (nine amino acids) is 35.29 kDa; the molecular mass calculated from the column run was 70.38 kDa. Arrows indicate the elution of your typical proteins in the gel filtration column. mAU: milli absorption units. (TIF) Figure S7 Enzyme activity of the ectoine hydroxylase just isn’t reversible. The forward and backward enzyme reactions of the EctD protein from S. alaskensis have been tested, as well as the formation of ectoine and hydroxyectoine was monitored by HPLC analysis. (a) Chromatograms from HPLC measurements monitored at 210 nm of a mixture of commercially available ectoine and 5-hydroxyectoine requirements. (b) HPLC tracing with the EctD-catalyzed enzyme reaction mixture that initially contained 6 mM ectoine; the enzyme assay was run for 20 min. (c) HPLC tracing from the EctD-catalyzed “reverse” enzyme reaction mixture that initially contained six mM hydroxyectoine; the enzyme assay was run for 24 h. (TIF) Figure S8 Phylogenetic tree of EctD-type proteins. The phylogenetic tree of ectoine hydroxylases shown is based on the alignment of EctD amino acid sequences identified by a BLAST search in the JGI Web-server, and that have been then aligned applying ClustalW. The phylogenetic distribution of your aligned EctD proteins was assessed by means of the iTOL Web-server. Evolutionary distances will not be given. The colour code indicates the distribution of EctD amongst members in the Bacteria and Archaea. (TIF) Table S1 Information collection and refinement statistics for the crystal structure with the EctD protein from V. salexigens in its iron-free kind. (DOC)Crystallization of your V. Salexigens EctD Protein in its Ironfree FormCrystallization trials have been performed employing the sitting-drop vapor diffusion technique at 20uC. A homogenous protein resolution on the affinity-purified EctD protein (in 20 mM TES pH 7.five, 80 mM NaCl) was concentrated to 10 mg/ml prior to crystallization experiments. EctD crystals have been grown by mixing 1.five ml protein option with 1.five ml reservoir solution containing 100 mM MES pH 5.0 and 1.two M ammonium sulfate; the EctD crystals grew inside 62 days to their final size of around 806906100 mm3.Canthaxanthin Reactive Oxygen Species Crystals have been cryoprotected by very carefully adding 1 ml 100 glycerol to the crystallization drop prior to freezing the crystals in liquid nitrogen.Lumichrome custom synthesis Data Collection, Refinement and Crystallographic Evaluation of the EctD ProteinEctD crystals diffracted X-rays to a minimum resolution of 1.PMID:23937941 85 A for the apo-EctD. The dataset was collected in the ID23-EH2 beamline in the ESRF (Grenoble, France) and processed with XDS [79]. The crystal structure of your iron-bound V. salexigens EctD protein (PDB code: 3EMR) [44] was applied as a template to acquire initial phases using PHASER [80]. The structure was further refined applying REFMAC5 [81] and manually adjusted using COOT [82]. Dataset and refinement statistics for the apoEctD crystal structure are listed in Table S1 and have been analyzed with Procheck [8.