Improving protein crystal quality by the without-oil microbatch method: crystallization and preliminary X-ray diffraction analysis of glutathione synthetase from Pseudoalteromonas haloplanktis.
Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from l-γ-glutamyl- l-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date.
Dipartimento di Chimica "Paolo Corradini", Università di Napoli Federico II, Complesso Universitario di Monte Sant'Angelo, Via Cinthia, Naples I-80126, Italy; E-Mails:  antonello.merlino@unina.it  (A.M.);  irene.russokrauss@unina.it  (I.R.K.);  andrea.pica@unina.it  (A.P.);  alessandro.vergara@unina.it  (A.V.).
Antonello
A
Merlino
Irene
I
Russo Krauss
Antonella
A
Albino
Andrea
A
Pica
Alessandro
A
Vergara
Mariorosario
M
Masullo
Emmanuele
E
De Vendittis
Filomena
F
Sica
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003460:Crystallization / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005981:Glutathione Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction / Q000379:methods*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005981:Glutathione Synthase