Overproduction and preliminary crystallographic study of a human kynurenine aminotransferase II homologue from Pyrococcus horikoshii OT3.
The Pyrococcus horikoshii OT3 genome contains a gene encoding a human kynurenine aminotransferase II (KAT II) homologue, which consists of 428 amino-acid residues and shows an amino-acid sequence identity of 30% to human KAT II. This gene was overexpressed in Escherichia coli and the recombinant protein (Ph-KAT II) was purified. Gel-filtration chromatography showed that Ph-KAT II exists as a homodimer. Ph-KAT II exhibited enzymatic activity that catalyzes the transamination of L-kynurenine to produce kynurenic acid. Crystals of Ph-KAT II were grown using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.2 A resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the centred orthorhombic space group C222(1), with unit-cell parameters a = 71.75, b = 86.84, c = 137.30 A. Assuming one molecule per asymmetric unit, the VM value was 2.19 A3 Da(-1) and the solvent content was 43.3%.
Chon
Hyongi
H
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Matsumura
Hiroyoshi
H
Shimizu
Shoko
S
Maeda
Nao
N
Koga
Yuichi
Y
Takano
Kazufumi
K
Kanaya
Shigenori
S
D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification; D001483:Base Sequence; D003460:Crystallization; D017931:DNA Primers; D020745:Genome, Archaeal; D006801:Humans; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014961:X-Ray Diffraction
D019843:Archaeal Proteins; D017931:DNA Primers; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase