Crystallization and preliminary X-ray analysis of 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-D-gluconate and NADPH.
NADPH-dependent 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-D-gluconate and D-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-D-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288 K. A crystal of the 5KGR-NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75 Å resolution. The crystal of the complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.6, c=62.9 Å. A crystal of the 5KGR-NADPH-5-keto-D-gluconate complex was prepared by soaking the 5KGR-NADPH complex crystal in reservoir solution supplemented with 100 mM 5-keto-D-gluconate and 10 mM NADPH for 20 min and diffracted X-rays to 2.26 Å resolution. The crystal of the ternary complex belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=128.7, c=62.5 Å. Both crystals contained two molecules in the asymmetric unit.
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.
Keiko
K
Kubota
Ken-ichi
K
Miyazono
Koji
K
Nagata
Hirohide
H
Toyama
Kazunobu
K
Matsushita
Masaru
M
Tanokura
D001426:Bacterial Proteins / Q000737:chemistry*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D005942:Gluconates / Q000737:chemistry*; D020576:Gluconobacter / Q000201:enzymology*; D009249:NADP / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*
D001426:Bacterial Proteins; D005942:Gluconates; D009249:NADP; D010088:Oxidoreductases; D002237:Carbohydrate Dehydrogenases; C093584:gluconate 5-dehydrogenase