Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis.
Genome analysis of Shewanella oneidensis, a Gram-negative bacterium with an unusual repertoire of respiratory and redox capabilities, revealed the presence of six glutathione S-transferase-like genes (sogst1-sogst6). Glutathione S-transferases (GSTs; EC 2.5.1.18) are found in all kingdoms of life and are involved in phase II detoxification processes by catalyzing the nucleophilic attack of reduced glutathione on diverse electrophilic substrates, thereby decreasing their reactivity. Structure-function studies of prokaryotic GST-like proteins are surprisingly underrepresented in the scientific literature when compared with eukaryotic GSTs. Here, the production and purification of recombinant SoGST3 (SO_1576) and SoGST6 (SO_4697), two of the six GST-like proteins in S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented. SoGST3 was crystallized in two different crystal forms in the presence of GSH and DTT that diffracted to high resolution: a primitive trigonal form in space group P3(1) that exhibited merohedral twinning with a high twin fraction and a primitive monoclinic form in space group P2(1). SoGST6 yielded primitive orthorhombic crystals in space group P2(1)2(1)2(1) from which diffraction data could be collected to medium resolution after application of cryo-annealing protocols. Crystal structures of both SoGST3 and SoGST6 have been determined based on marginal search models by maximum-likelihood molecular replacement as implemented in the program Phaser.
Remmerie
Bert
B
Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), K. L. Ledeganckstraat, Ghent University, B-9000 Ghent, Belgium.
Vandenbroucke
Karen
K
De Smet
Lina
L
Carpentier
Wesley
W
De Vos
Dirk
D
Stout
Jan
J
Van Beeumen
Jozef
J
Savvides
Savvas N
SN
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology*; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione