Abstract
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.