title: Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase.
abstract: Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the beta-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 A resolution, are of the protein in complex with Mn2+-GDP.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Joyce
authors.forename: Edward R
authors.initials: ER
authors.lastname: Brownie
authors.forename: Koto
authors.initials: K
authors.lastname: Hayakawa
authors.forename: Marie E
authors.initials: ME
authors.lastname: Fraser
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry*; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D013824:Thermus / Q000201:enzymology*
chemical_list: D000214:Acyl Coenzyme A; D017931:DNA Primers; C012046:succinyl-coenzyme A; D008025:Ligases