Purification, crystallization and preliminary X-ray analysis of L-sorbose reductase from Gluconobacter frateurii complexed with L-sorbose or NADPH.
NADPH-dependent L-sorbose reductase (SR) from Gluconobacter frateurii was expressed in Escherichia coli, purified and crystallized with L-sorbose or NADPH using the sitting-drop vapour-diffusion method at 293 K. Crystals of the SR-L-sorbose complex and the SR-NADPH complex were obtained using reservoir solutions containing PEG 2000 or PEG 400 as precipitants and diffracted X-rays to 2.38 and 1.90 A resolution, respectively. The crystal of the SR-L-sorbose complex belonged to space group C222(1), with unit-cell parameters a = 124.2, b = 124.1, c = 60.8 A. The crystal of the SR-NADPH complex belonged to space group P2(1), with unit-cell parameters a = 124.3, b = 61.0, c = 124.5 A, beta = 89.99 degrees . The crystals contained two and eight molecules, respectively, in the asymmetric unit.
Kubota
Keiko
K
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Nagata
Koji
K
Miyazono
Ken-ichi
K
Toyama
Hirohide
H
Matsushita
Kazunobu
K
Tanokura
Masaru
M
D003001:Cloning, Molecular; D003460:Crystallization; D003625:Data Collection; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D020576:Gluconobacter / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*; D013013:Sorbose / Q000378:metabolism*; D013223:Statistics as Topic; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature; D014961:X-Ray Diffraction
D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; C506437:sorbose reductase; D013013:Sorbose