Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii.
Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.
Inagaki
Eiji
E
RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Sakamoto
Keiko
K
Obayashi
Naomi
N
Terada
Takaho
T
Shirouzu
Mikako
M
Bessho
Yoshitaka
Y
Kuroishi
Chizu
C
Kuramitsu
Seiki
S
Shinkai
Akeo
A
Yokoyama
Shigeyuki
S
D001051:Apoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005686:Galactokinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D019848:Gene Expression Regulation, Archaeal; D007700:Kinetics; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D001051:Apoenzymes; D019843:Archaeal Proteins; D011994:Recombinant Proteins; D005686:Galactokinase