Expression, purification, crystallization and preliminary X-ray analysis of the Met244Ala variant of catalase-peroxidase (KatG) from the haloarchaeon Haloarcula marismortui.
The covalent modification of the side chains of Trp95, Tyr218 and Met244 within the active site of Haloarcula marismortui catalase-peroxidase (KatG) appears to be common to all KatGs and has been demonstrated to be particularly significant for its bifunctionality [Smulevich et al. (2006), J. Inorg. Biochem. 100, 568-585; Jakopitsch, Kolarich et al. (2003), FEBS Lett. 552, 135-140; Jakopitsch, Auer et al. (2003), J. Biol. Chem. 278, 20185-20191; Jakopitsch et al. (2004), J. Biol. Chem. 279, 46082-46095; Regelsberger et al. (2001), Biochem. Soc. Trans. 29, 99-105; Ghiladi, Knudsen et al. (2005), J. Biol. Chem. 280, 22651-22663; Ghiladi, Medzihradzky et al. (2005), Biochemistry, 44, 15093-15105]. The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant showed a complete loss of catalase activity, whereas the peroxidase activity of this mutant was highly enhanced owing to an increase in its affinity for the peroxidatic substrate. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 A, beta = 99.81 degrees . A crystal frozen using lithium sulfate as the cryoprotectant diffracted to beyond 2.0 A resolution. Preliminary X-ray analysis suggests the presence of a dimer in the asymmetric unit.
Ten-I
Tomomi
T
Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-10 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Kumasaka
Takashi
T
Higuchi
Wataru
W
Tanaka
Satoru
S
Yoshimatsu
Katsuhiko
K
Fujiwara
Taketomo
T
Sato
Takao
T
D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D007700:Kinetics; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; C118175:catalase-peroxidase, Haloarcula